https://proteopedia.org/api.php?action=feedcontributions&feedformat=atom&user=Autumn+Forrester 2026-09-16T18:12:32Z User contributions MediaWiki 1.43.8 https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550970 2022-04-26T13:44:47Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt.pdb' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/5'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp&lt;ref&gt;National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].&lt;/ref&gt;, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |600px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam&lt;ref&gt;Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913&lt;/ref&gt; to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |800px| center | thumb | Pfam query aligned with a cytidylate kinase-like family.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |700px| center | thumb | Top domain results from Pfam.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI&lt;ref&gt;Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.&lt;/ref&gt; query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 400px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> We used POCASA &lt;ref&gt;J. Yu, Y. Zhou, I. Tanaka, M. Yao, Roll: A new algorithm for the detection of protein pockets and cavities with a rolling probe sphere. Bioinformatics, 26(1), 46-52, (2010) [PMID: 19846440]&lt;/ref&gt; to determine potential binding pockets within our protein and PyRx&lt;ref&gt;Small-Molecule Library Screening by Docking with PyRx. Dallakyan S, Olson AJ. Methods Mol Biol. 2015;1263:243-50.&lt;/ref&gt; to actually bind dCMP to 3HDT. Then PyMOL was used to visualize the binding pockets and dCMP in the protein. This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; (in purple) is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. However, this area was where dCMP binded with the highest affinity in PyRx. The &lt;scene name='90/904996/Hydrophobic_interactions/1'&gt;amino acids&lt;/scene&gt; (Gly21, Ser22, Gly23, Val27, Thr 142, Gln149, Arg150, Thr197, Leu200, Thr201) interact within that area to help hold the substrate in place in the protein with hydrophobic interactions. Also, there are two residues, &lt;scene name='90/904996/Dcmp_with_3hdt_active_site/1'&gt;Lys146 (purple) and Leu 202 (yellow)&lt;/scene&gt; within the active site containing dCMP and cofactor ATP that are important in substrate binding. They interact with the substrate forming hydrogen bonds with the oxygens on the phosphate groups and the 6-membered ring. However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful in getting the program to recognize where the ATP was bound in the protein first before docking our substrate with ATP already in the protein.<br /> <br /> [[Image:POCASA 3hdt image 2.png | 500px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling.]] <br /> Some of the predicted bind pockets were areas where ATP (red arrow) and dCMP (yellow arrow) binded to with the highest affinity in PyRx.<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink). Visualized in PyMOL&lt;ref&gt;The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.&lt;/ref&gt;. ]]<br /> [[Image:DCMP reaction.png |275px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> <br /> <br /> == Laboratory Experiments ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT.]]<br /> <br /> [[Image:KineticstableJRAF2022.PNG‎ |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT.]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT.]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550969 2022-04-26T13:44:24Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt.pdb' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/5'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp&lt;ref&gt;National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].&lt;/ref&gt;, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |600px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam&lt;ref&gt;Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913&lt;/ref&gt; to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |800px| center | thumb | Pfam query aligned with a cytidylate kinase-like family.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |700px| center | thumb | Top domain results from Pfam.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI&lt;ref&gt;Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.&lt;/ref&gt; query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 400px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> We used POCASA &lt;ref&gt;J. Yu, Y. Zhou, I. Tanaka, M. Yao, Roll: A new algorithm for the detection of protein pockets and cavities with a rolling probe sphere. Bioinformatics, 26(1), 46-52, (2010) [PMID: 19846440]&lt;/ref&gt; to determine potential binding pockets within our protein and PyRx&lt;ref&gt;Small-Molecule Library Screening by Docking with PyRx. Dallakyan S, Olson AJ. Methods Mol Biol. 2015;1263:243-50.&lt;/ref&gt; to actually bind dCMP to 3HDT. Then PyMOL was used to visualize the binding pockets and dCMP in the protein. This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; (in purple) is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. However, this area was where dCMP binded with the highest affinity in PyRx. The &lt;scene name='90/904996/Hydrophobic_interactions/1'&gt;amino acids&lt;/scene&gt; (Gly21, Ser22, Gly23, Val27, Thr 142, Gln149, Arg150, Thr197, Leu200, Thr201) interact within that area to help hold the substrate in place in the protein with hydrophobic interactions. Also, there are two residues, &lt;scene name='90/904996/Dcmp_with_3hdt_active_site/1'&gt;Lys146 (purple) and Leu 202 (yellow)&lt;/scene&gt; within the active site containing dCMP and cofactor ATP that are important in substrate binding. They interact with the substrate forming hydrogen bonds with the oxygens on the phosphate groups and the 6-membered ring. However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful in getting the program to recognize where the ATP was bound in the protein first before docking our substrate with ATP already in the protein.<br /> <br /> [[Image:POCASA 3hdt image 2.png | 500px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling.]] <br /> Some of the predicted bind pockets were areas where ATP (red arrow) and dCMP (yellow arrow) binded to with the highest affinity in PyRx.<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink). Visualized in PyMOL&lt;ref&gt;The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.&lt;/ref&gt;. ]]<br /> [[Image:DCMP reaction.png |300px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> <br /> <br /> == Laboratory Experiments ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT.]]<br /> <br /> [[Image:KineticstableJRAF2022.PNG‎ |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT.]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT.]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550962 2022-04-26T05:13:06Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt.pdb' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/5'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp&lt;ref&gt;National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].&lt;/ref&gt;, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |600px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam&lt;ref&gt;Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913&lt;/ref&gt; to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |800px| center | thumb | Pfam query aligned with a cytidylate kinase-like family.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |700px| center | thumb | Top domain results from Pfam.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI&lt;ref&gt;Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.&lt;/ref&gt; query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 400px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> We used POCASA &lt;ref&gt;J. Yu, Y. Zhou, I. Tanaka, M. Yao, Roll: A new algorithm for the detection of protein pockets and cavities with a rolling probe sphere. Bioinformatics, 26(1), 46-52, (2010) [PMID: 19846440]&lt;/ref&gt; to determine potential binding pockets within our protein and PyRx&lt;ref&gt;Small-Molecule Library Screening by Docking with PyRx. Dallakyan S, Olson AJ. Methods Mol Biol. 2015;1263:243-50.&lt;/ref&gt; to actually bind dCMP to 3HDT. Then PyMOL was used to visualize the binding pockets and dCMP in the protein. This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; (in purple) is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. However, this area was where dCMP binded with the highest affinity in PyRx. The &lt;scene name='90/904996/Hydrophobic_interactions/1'&gt;amino acids&lt;/scene&gt; (Gly21, Ser22, Gly23, Val27, Thr 142, Gln149, Arg150, Thr197, Leu200, Thr201) interact within that area to help hold the substrate in place in the protein with hydrophobic interactions. Also, there are two residues, &lt;scene name='90/904996/Dcmp_with_3hdt_active_site/1'&gt;Lys146 (purple) and Leu 202 (yellow)&lt;/scene&gt; within the active site containing dCMP and cofactor ATP that are important in substrate binding. They interact with the substrate forming hydrogen bonds with the oxygens on the phosphate groups and the 6-membered ring. However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful in getting the program to recognize where the ATP was bound in the protein first before docking our substrate with ATP already in the protein.<br /> <br /> [[Image:POCASA 3hdt image 2.png | 500px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling.]] <br /> Some of the predicted bind pockets were areas where ATP (red arrow) and dCMP (yellow arrow) binded to with the highest affinity in PyRx.<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink). Visualized in PyMOL&lt;ref&gt;The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.&lt;/ref&gt;. ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> <br /> <br /> == Laboratory Experiments ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550960 2022-04-26T05:12:34Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt.pdb' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/5'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp&lt;ref&gt;National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].&lt;/ref&gt;, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |600px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam&lt;ref&gt;Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913&lt;/ref&gt; to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |800px| center | thumb | Pfam query aligned with a cytidylate kinase-like family.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |700px| center | thumb | Top domain results from Pfam.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI&lt;ref&gt;Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.&lt;/ref&gt; query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 400px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> We used POCASA &lt;ref&gt;J. Yu, Y. Zhou, I. Tanaka, M. Yao, Roll: A new algorithm for the detection of protein pockets and cavities with a rolling probe sphere. Bioinformatics, 26(1), 46-52, (2010) [PMID: 19846440]&lt;/ref&gt; to determine potential binding pockets within our protein and PyRx&lt;ref&gt;Small-Molecule Library Screening by Docking with PyRx. Dallakyan S, Olson AJ. Methods Mol Biol. 2015;1263:243-50.&lt;/ref&gt; to actually bind dCMP to 3HDT. Then PyMOL was used to visualize the binding pockets and dCMP in the protein. This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; (in purple) is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. However, this area was where dCMP binded with the highest affinity in PyRx. The &lt;scene name='90/904996/Hydrophobic_interactions/1'&gt;amino acids&lt;/scene&gt; (Gly21, Ser22, Gly23, Val27, Thr 142, Gln149, Arg150, Thr197, Leu200, Thr201) interact within that area to help hold the substrate in place in the protein with hydrophobic interactions. Also, there are two residues, &lt;scene name='90/904996/Dcmp_with_3hdt_active_site/1'&gt;Lys146 (purple) and Leu 202 (yellow)&lt;/scene&gt; within the active site containing dCMP and cofactor ATP that are important in substrate binding. They interact with the substrate forming hydrogen bonds with the oxygens on the phosphate groups and the 6-membered ring. However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful in getting the program to recognize where the ATP was bound in the protein first before docking our substrate with ATP already in the protein.<br /> <br /> [[Image:POCASA 3hdt image 2.png | 500px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling.]] <br /> Some of the predicted bind pockets were areas where ATP (red arrow) and dCMP (yellow arrow) binded to with the highest affinity in PyRx.<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink). Visualized in PyMOL&lt;ref&gt;The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.&lt;/ref&gt;. ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> == Laboratory Experiments ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550959 2022-04-26T05:12:06Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt.pdb' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/5'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp&lt;ref&gt;National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].&lt;/ref&gt;, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |600px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam&lt;ref&gt;Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913&lt;/ref&gt; to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |800px| center | thumb | Pfam query aligned with a cytidylate kinase-like family.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |700px| center | thumb | Top domain results from Pfam.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI&lt;ref&gt;Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.&lt;/ref&gt; query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 400px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> We used POCASA &lt;ref&gt;J. Yu, Y. Zhou, I. Tanaka, M. Yao, Roll: A new algorithm for the detection of protein pockets and cavities with a rolling probe sphere. Bioinformatics, 26(1), 46-52, (2010) [PMID: 19846440]&lt;/ref&gt; to determine potential binding pockets within our protein and PyRx&lt;ref&gt;Small-Molecule Library Screening by Docking with PyRx. Dallakyan S, Olson AJ. Methods Mol Biol. 2015;1263:243-50.&lt;/ref&gt; to actually bind dCMP to 3HDT. Then PyMOL was used to visualize the binding pockets and dCMP in the protein. This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; (in purple) is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. However, this area was where dCMP binded with the highest affinity in PyRx. The &lt;scene name='90/904996/Hydrophobic_interactions/1'&gt;amino acids&lt;/scene&gt; (Gly21, Ser22, Gly23, Val27, Thr 142, Gln149, Arg150, Thr197, Leu200, Thr201) interact within that area to help hold the substrate in place in the protein with hydrophobic interactions. Also, there are two residues, &lt;scene name='90/904996/Dcmp_with_3hdt_active_site/1'&gt;Lys146 (yellow) and Leu 202 (purple)&lt;/scene&gt; within the active site containing dCMP and cofactor ATP that are important in substrate binding. They interact with the substrate forming hydrogen bonds with the oxygens on the phosphate groups and the 6-membered ring. However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful in getting the program to recognize where the ATP was bound in the protein first before docking our substrate with ATP already in the protein.<br /> <br /> [[Image:POCASA 3hdt image 2.png | 500px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling.]] <br /> Some of the predicted bind pockets were areas where ATP (red arrow) and dCMP (yellow arrow) binded to with the highest affinity in PyRx.<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink). Visualized in PyMOL&lt;ref&gt;The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.&lt;/ref&gt;. ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> == Laboratory Experiments ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550957 2022-04-26T05:11:11Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt.pdb' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/5'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp&lt;ref&gt;National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].&lt;/ref&gt;, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |600px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam&lt;ref&gt;Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913&lt;/ref&gt; to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |800px| center | thumb | Pfam query aligned with a cytidylate kinase-like family.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |700px| center | thumb | Top domain results from Pfam.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI&lt;ref&gt;Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.&lt;/ref&gt; query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 400px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> We used POCASA &lt;ref&gt;J. Yu, Y. Zhou, I. Tanaka, M. Yao, Roll: A new algorithm for the detection of protein pockets and cavities with a rolling probe sphere. Bioinformatics, 26(1), 46-52, (2010) [PMID: 19846440]&lt;/ref&gt; to determine potential binding pockets within our protein and PyRx&lt;ref&gt;Small-Molecule Library Screening by Docking with PyRx. Dallakyan S, Olson AJ. Methods Mol Biol. 2015;1263:243-50.&lt;/ref&gt; to actually bind dCMP to 3HDT. Then PyMOL was used to visualize the binding pockets and dCMP in the protein. This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; (in purple) is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. However, this area was where dCMP binded with the highest affinity in PyRx. The &lt;scene name='90/904996/Hydrophobic_interactions/1'&gt;amino acids&lt;/scene&gt; (Gly21, Ser22, Gly23, Val27, Thr 142, Gln149, Arg150, Thr197, Leu200, Thr201) interact within that area to help hold the substrate in place in the protein with hydrophobic interactions. Also, there are two residues, &lt;scene name='90/904996/Dcmp_with_3hdt_active_site/1'&gt;Lys146 and Leu 202&lt;/scene&gt; within the active site containing dCMP and cofactor ATP that are important in substrate binding. They interact with the substrate forming hydrogen bonds with the oxygens on the phosphate groups and the 6-membered ring. However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful in getting the program to recognize where the ATP was bound in the protein first before docking our substrate with ATP already in the protein.<br /> <br /> [[Image:POCASA 3hdt image 2.png | 500px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling.]] <br /> Some of the predicted bind pockets were areas where ATP (red arrow) and dCMP (yellow arrow) binded to with the highest affinity in PyRx.<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink). Visualized in PyMOL&lt;ref&gt;The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.&lt;/ref&gt;. ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> == Laboratory Experiments ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550955 2022-04-26T04:45:03Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt.pdb' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/5'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp&lt;ref&gt;National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].&lt;/ref&gt;, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |600px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam&lt;ref&gt;Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913&lt;/ref&gt; to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |800px| center | thumb | Pfam query aligned with a cytidylate kinase-like family.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |700px| center | thumb | Top domain results from Pfam.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI&lt;ref&gt;Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.&lt;/ref&gt; query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 400px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> We used POCASA &lt;ref&gt;J. Yu, Y. Zhou, I. Tanaka, M. Yao, Roll: A new algorithm for the detection of protein pockets and cavities with a rolling probe sphere. Bioinformatics, 26(1), 46-52, (2010) [PMID: 19846440]&lt;/ref&gt; to determine potential binding pockets within our protein and PyRx&lt;ref&gt;Small-Molecule Library Screening by Docking with PyRx. Dallakyan S, Olson AJ. Methods Mol Biol. 2015;1263:243-50.&lt;/ref&gt; to actually bind dCMP to 3HDT. Then PyMOL was used to visualize the binding pockets and dCMP in the protein. This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; (in purple) is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. However, this area was where dCMP binded with the highest affinity in PyRx. The &lt;scene name='90/904996/Hydrophobic_interactions/1'&gt;amino acids&lt;/scene&gt; (Gly21, Ser22, Gly23, Val27, Thr 142, Gln149, Arg150, Thr197, Leu200, Thr201) interact within that area to help hold the substrate in place in the protein with hydrophobic interactions. Also, there are two amino acids &lt;scene name='90/904996/H-bonds/1'&gt;Lys146 and Leu 202&lt;/scene&gt; that interact with dCMP with hydrogen bonds. However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful in getting the program to recognize where the ATP was bound in the protein first before docking our substrate with ATP already in the protein.<br /> <br /> [[Image:POCASA 3hdt image 2.png | 500px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling.]] <br /> Some of the predicted bind pockets were areas where ATP (red arrow) and dCMP (yellow arrow) binded to with the highest affinity in PyRx.<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink). Visualized in PyMOL&lt;ref&gt;The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.&lt;/ref&gt;. ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> <br /> <br /> Active site containing dCMP and cofactor ATP can be found &lt;scene name='90/904996/Dcmp_with_3hdt_active_site/1'&gt;here&lt;/scene&gt; with residues important in substrate binding highlighted in purple (Lys146) and yellow (Leu202).<br /> <br /> == Laboratory Experiments ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550954 2022-04-26T04:38:49Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt.pdb' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/5'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp&lt;ref&gt;National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].&lt;/ref&gt;, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |600px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam&lt;ref&gt;Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913&lt;/ref&gt; to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |800px| center | thumb | Pfam query aligned with a cytidylate kinase-like family.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |700px| center | thumb | Top domain results from Pfam.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI&lt;ref&gt;Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.&lt;/ref&gt; query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 400px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> We used POCASA to determine potential binding pockets within our protein and PyRx&lt;ref&gt;Small-Molecule Library Screening by Docking with PyRx. Dallakyan S, Olson AJ. Methods Mol Biol. 2015;1263:243-50.&lt;/ref&gt; to actually bind dCMP to 3HDT. Then PyMOL was used to visualize the binding pockets and dCMP in the protein. This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; (in purple) is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. However, this area was where dCMP binded with the highest affinity in PyRx. The &lt;scene name='90/904996/Hydrophobic_interactions/1'&gt;amino acids&lt;/scene&gt; (Gly21, Ser22, Gly23, Val27, Thr 142, Gln149, Arg150, Thr197, Leu200, Thr201) interact within that area to help hold the substrate in place in the protein with hydrophobic interactions. Also, there are two amino acids &lt;scene name='90/904996/H-bonds/1'&gt;Lys146 and Leu 202&lt;/scene&gt; that interact with dCMP with hydrogen bonds. However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful in getting the program to recognize where the ATP was bound in the protein first before docking our substrate with ATP already in the protein.<br /> <br /> [[Image:POCASA 3hdt image 2.png | 500px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling.]] <br /> Some of the predicted bind pockets were areas where ATP (red arrow) and dCMP (yellow arrow) binded to with the highest affinity in PyRx.<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink). Visualized in PyMOL&lt;ref&gt;The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.&lt;/ref&gt;. ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> <br /> <br /> Active site containing dCMP and cofactor ATP can be found &lt;scene name='90/904996/Dcmp_with_3hdt_active_site/1'&gt;here&lt;/scene&gt; with residues important in substrate binding highlighted in purple (Lys146) and yellow (Leu202).<br /> <br /> == Laboratory Experiments ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550953 2022-04-26T04:37:31Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt.pdb' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/5'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp&lt;ref&gt;National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].&lt;/ref&gt;, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |600px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam&lt;ref&gt;Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913&lt;/ref&gt; to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |800px| center | thumb | Pfam query aligned with a cytidylate kinase-like family.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |700px| center | thumb | Top domain results from Pfam.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI&lt;ref&gt;Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.&lt;/ref&gt; query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 400px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> We used POCASA to determine potential binding pockets within our protein and PyRx&lt;ref&gt;Small-Molecule Library Screening by Docking with PyRx. Dallakyan S, Olson AJ. Methods Mol Biol. 2015;1263:243-50.&lt;/ref&gt; to actually bind dCMP to 3HDT. Then PyMOL was used to visualize the binding pockets and dCMP in the protein. This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; (in purple) is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. However, this area was where dCMP binded with the highest affinity in PyRx. The &lt;scene name='90/904996/Hydrophobic_interactions/1'&gt;amino acids&lt;/scene&gt; (Gly21, Ser22, Gly23, Val27, Thr 142, Gln149, Arg150, Thr197, Leu200, Thr201) interact within that area to help hold the substrate in place in the protein with hydrophobic interactions. Also, there are two amino acids &lt;scene name='90/904996/H-bonds/1'&gt;Lys146 and Leu 202&lt;/scene&gt; that interact with dCMP with hydrogen bonds. However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful in getting the program to recognize where the ATP was bonded in the protein when docking our substrate.<br /> <br /> [[Image:POCASA 3hdt image 2.png | 500px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling.]] <br /> Some of the predicted bind pockets were areas where ATP (red arrow) and dCMP (yellow arrow) binded to with the highest affinity in PyRx.<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink). Visualized in PyMOL&lt;ref&gt;The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.&lt;/ref&gt;. ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> <br /> <br /> Active site containing dCMP and cofactor ATP can be found &lt;scene name='90/904996/Dcmp_with_3hdt_active_site/1'&gt;here&lt;/scene&gt; with residues important in substrate binding highlighted in purple (Lys146) and yellow (Leu202).<br /> <br /> == Laboratory Experiments ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550952 2022-04-26T04:32:16Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt.pdb' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/5'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp&lt;ref&gt;National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].&lt;/ref&gt;, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |600px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam&lt;ref&gt;Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913&lt;/ref&gt; to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |800px| center | thumb | Pfam query aligned with a cytidylate kinase-like family.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |700px| center | thumb | Top domain results from Pfam.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI&lt;ref&gt;Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.&lt;/ref&gt; query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 400px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> We used POCASA to determine potential binding pockets within our protein and PyRx&lt;ref&gt;Small-Molecule Library Screening by Docking with PyRx. Dallakyan S, Olson AJ. Methods Mol Biol. 2015;1263:243-50.&lt;/ref&gt; to actually bind dCMP to 3HDT. Then PyMOL was used to visualize the binding pockets and dCMP in the protein. This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; (in purple) is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. However, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are &lt;scene name='90/904996/H-bonds/1'&gt;Lys146 and Leu 202&lt;/scene&gt; which interact with dCMP by hydrogen bonds. Also, there are other &lt;scene name='90/904996/Hydrophobic_interactions/1'&gt;amino acids&lt;/scene&gt; (Gly21, Ser22, Gly23, Val27, Thr 142, Gln149, Arg150, Thr197, Leu200, Thr201) that help hold the substrate in place in the protein. <br /> However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful <br /> <br /> [[Image:POCASA 3hdt image 2.png | 500px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling.]] <br /> Some of the predicted bind pockets were areas where ATP (red arrow) and dCMP (yellow arrow) binded to with the highest affinity in PyRx.<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink). Visualized in PyMOL&lt;ref&gt;The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.&lt;/ref&gt;. ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> <br /> <br /> Active site containing dCMP and cofactor ATP can be found &lt;scene name='90/904996/Dcmp_with_3hdt_active_site/1'&gt;here&lt;/scene&gt; with residues important in substrate binding highlighted in purple (Lys146) and yellow (Leu202).<br /> <br /> == Laboratory Experiments ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550951 2022-04-26T04:26:31Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt.pdb' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/5'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp&lt;ref&gt;National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].&lt;/ref&gt;, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |600px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam&lt;ref&gt;Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913&lt;/ref&gt; to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |800px| center | thumb | Pfam query aligned with a cytidylate kinase-like family.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |700px| center | thumb | Top domain results from Pfam.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI&lt;ref&gt;Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.&lt;/ref&gt; query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 400px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> We used POCASA to determine potential binding pockets within our protein and PyRx&lt;ref&gt;Small-Molecule Library Screening by Docking with PyRx. Dallakyan S, Olson AJ. Methods Mol Biol. 2015;1263:243-50.&lt;/ref&gt; to actually bind dCMP to 3HDT. Then PyMOL was used to visualize the binding pockets and dCMP in the protein. This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; (in purple) is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. However, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> Also, there are other &lt;scene name='90/904996/Hydrophobic_interactions/1'&gt;amino acids&lt;/scene&gt; (Gly21, Ser22, Gly23, Val27, Thr 142, Gln149, Arg150, Thr197, Leu200, Thr201) that help hold the substrate in place in the protein. <br /> However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful <br /> <br /> [[Image:POCASA 3hdt image 2.png | 500px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling.]] <br /> Some of the predicted bind pockets were areas where ATP (red arrow) and dCMP (yellow arrow) binded to with the highest affinity in PyRx.<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink). Visualized in PyMOL&lt;ref&gt;The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.&lt;/ref&gt;. ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> <br /> <br /> Active site containing dCMP and cofactor ATP can be found &lt;scene name='90/904996/Dcmp_with_3hdt_active_site/1'&gt;here&lt;/scene&gt; with residues important in substrate binding highlighted in purple (Lys146) and yellow (Leu202).<br /> <br /> == Laboratory Experiments ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550930 2022-04-26T03:37:42Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt.pdb' size='340' side='right' caption='Structure of putative kinase 3HDT' &lt;scene name='90/904996/Dcmp_with_3hdtintro/1'&gt;scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;&lt;/scene&gt;&lt;scene name='90/904996/Dcmp_with_3hdtintro/1'&gt;3HDT with dCMP and ATP docked.&lt;/scene&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |600px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |800px| center | thumb | Pfam query aligned with a cytidylate kinase-like family.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |700px| center | thumb | Top domain results from Pfam.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 400px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> We used POCASA to determine potential binding pockets within our protein and PyRX to actually bind dCMP to 3HDT. Then PyMOL was used to visualize the binding pockets and dCMP in the protein. This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; (in purple) is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. However, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful <br /> <br /> [[Image:POCASA 3hdt image 2.png | 500px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling.]] <br /> Some of the predicted binding pockets were areas where ATP (red arrow) and dCMP (yellow arrow) binded to with the highest affinity in PyRx. This image is only showing one subunit of 3hdt.<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> &lt;scene name='90/904996/Dcmp_with_3hdt/1'&gt;dCMP and cofactor ATP docked in 3HDT&lt;/scene&gt;<br /> <br /> == Laboratory Experiments ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.<br /> <br /> 5. Small-Molecule Library Screening by Docking with PyRx. Dallakyan S, Olson AJ. Methods Mol Biol. 2015;1263:243-50.</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550928 2022-04-26T03:36:43Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt.pdb' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |600px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |800px| center | thumb | Pfam query aligned with a cytidylate kinase-like family.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |700px| center | thumb | Top domain results from Pfam.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 400px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> We used POCASA to determine potential binding pockets within our protein and PyRX to actually bind dCMP to 3HDT. Then PyMOL was used to visualize the binding pockets and dCMP in the protein. This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; (in purple) is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. However, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful <br /> <br /> [[Image:POCASA 3hdt image 2.png | 500px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling.]] <br /> Some of the predicted bind pockets were areas where ATP (red arrow) and dCMP (yellow arrow) binded to with the highest affinity in PyRx.<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> &lt;scene name='90/904996/Dcmp_with_3hdt/1'&gt;dCMP and cofactor ATP docked in 3HDT&lt;/scene&gt;<br /> <br /> == Laboratory Experiments ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.<br /> <br /> 5. Small-Molecule Library Screening by Docking with PyRx. Dallakyan S, Olson AJ. Methods Mol Biol. 2015;1263:243-50.</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550927 2022-04-26T03:34:54Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt.pdb' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |600px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |800px| center | thumb | Pfam query aligned with a cytidylate kinase-like family.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |700px| center | thumb | Top domain results from Pfam.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 400px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> We used POCASA to determine potential binding pockets within our protein and PyRX to actually bind dCMP to 3HDT. Then PyMOL was used to visualize the binding pockets and dCMP in the protein. This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; (in purple) is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. However, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful <br /> <br /> [[Image:POCASA 3hdt image 2.png | 500px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling.]] <br /> Some of the predicted bind pockets were areas where ATP (red arrow) and dCMP (yellow arrow) binded to with the highest affinity in PyRx.<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> &lt;scene name='90/904996/Dcmp_with_3hdt/1'&gt;dCMP and cofactor ATP docked in 3HDT&lt;/scene&gt;<br /> <br /> == Laboratory Experiments or ''In vitro'' analysis ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.<br /> <br /> 5. Small-Molecule Library Screening by Docking with PyRx. Dallakyan S, Olson AJ. Methods Mol Biol. 2015;1263:243-50.</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550926 2022-04-26T03:34:19Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt.pdb' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |600px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |800px| center | thumb | Pfam query aligned with a cytidylate kinase-like family.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |700px| center | thumb | Top domain results from Pfam.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 400px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> We used POCASA to determine potential binding pockets within our protein and PyRX to actually bind dCMP to 3HDT. Then PyMOL was used to visualize the binding pockets and dCMP in the protein. This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; (in purple) is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. However, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful <br /> <br /> [[Image:POCASA 3hdt image 2.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling.]] <br /> Some of the predicted bind pockets were areas where ATP (red arrow) and dCMP (yellow arrow) binded to with the highest affinity in PyRx.<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> &lt;scene name='90/904996/Dcmp_with_3hdt/1'&gt;dCMP and cofactor ATP docked in 3HDT&lt;/scene&gt;<br /> <br /> == Laboratory Experiments or ''In vitro'' analysis ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.<br /> <br /> 5. Small-Molecule Library Screening by Docking with PyRx. Dallakyan S, Olson AJ. Methods Mol Biol. 2015;1263:243-50.</div>

Autumn Forrester https://proteopedia.org/index.php?title=File:POCASA_3hdt_image_2.png&diff=3550925 2022-04-26T03:33:44Z

<p>Autumn Forrester: Predicted binding pockets for 3HDT from POCASA</p> <hr /> <div>Predicted binding pockets for 3HDT from POCASA</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550921 2022-04-26T03:27:28Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |600px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |800px| center | thumb | Pfam query aligned with a cytidylate kinase-like family.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |700px| center | thumb | Top domain results from Pfam.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 400px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> We used POCASA to determine potential binding pockets within our protein and PyRX to actually bind dCMP to 3HDT. Then PyMOL was used to visualize the binding pockets and dCMP in the protein. This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; (in purple) is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. However, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful <br /> <br /> [[Image:POCASA 3hdt .png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling.]] <br /> Some of the predicted bind pockets were areas where ATP (red arrow) and dCMP (yellow arrow) binded to with the highest affinity in PyRx.<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> &lt;scene name='90/904996/Dcmp_with_3hdt/1'&gt;dCMP and cofactor ATP docked in 3HDT&lt;/scene&gt;<br /> <br /> == Laboratory Experiments or ''In vitro'' analysis ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester https://proteopedia.org/index.php?title=File:POCASA_3hdt_.png&diff=3550916 2022-04-26T03:20:07Z

<p>Autumn Forrester: Predicted binding pockets for 3HDT from POCASA</p> <hr /> <div>Predicted binding pockets for 3HDT from POCASA</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550915 2022-04-26T03:19:00Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |600px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |800px| center | thumb | Pfam query aligned with a cytidylate kinase-like family.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |700px| center | thumb | Top domain results from Pfam.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 400px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> We used POCASA to determine potential binding pockets within our protein and PyRX to actually bind dCMP to 3HDT. Then PyMOL was used to visualize the binding pockets and dCMP in the protein. This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; (in purple) is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. However, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful <br /> <br /> [[Image:Possible binding pockets.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling ]]<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> == Laboratory Experiments or ''In vitro'' analysis ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550914 2022-04-26T03:16:44Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |600px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |800px| center | thumb | Pfam query aligned with a cytidylate kinase-like family.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |700px| center | thumb | Top domain results from Pfam.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 400px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> We used POCASA to determine potential binding pockets within our protein and PyRX to actually bind dCMP to 3HDT. Then PyMOL was used to visualize the binding pockets and dCMP in the protein. This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; is is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. Also, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful <br /> <br /> [[Image:Possible binding pockets.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling ]]<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> == Laboratory Experiments or ''In vitro'' analysis ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550891 2022-04-26T02:39:42Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |600px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |800px| center | thumb | Pfam query aligned with a cytidylate kinase-like family.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |700px| center | thumb | Top domain results from Pfam.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 400px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; is is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. Also, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful <br /> <br /> [[Image:Possible binding pockets.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling ]]<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> == Laboratory Experiments or ''In vitro'' analysis ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550890 2022-04-26T02:39:26Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |600px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |800px| center | thumb | Pfam query aligned with a cytidylate kinase-like family.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |800px| center | thumb | Top domain results from Pfam.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 400px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; is is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. Also, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful <br /> <br /> [[Image:Possible binding pockets.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling ]]<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> == Laboratory Experiments or ''In vitro'' analysis ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550889 2022-04-26T02:38:42Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |600px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |900px| center | thumb | Pfam query aligned with a cytidylate kinase-like family.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |900px| center | thumb | Top domain results from Pfam.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 400px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; is is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. Also, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful <br /> <br /> [[Image:Possible binding pockets.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling ]]<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> == Laboratory Experiments or ''In vitro'' analysis ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550888 2022-04-26T02:37:54Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |600px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |1000px| center | thumb | Pfam query aligned with a cytidylate kinase-like family.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |1000px| center | thumb | Top domain results from Pfam.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 400px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; is is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. Also, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful <br /> <br /> [[Image:Possible binding pockets.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling ]]<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> == Laboratory Experiments or ''In vitro'' analysis ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550887 2022-04-26T02:37:07Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |600px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |1100px| center | thumb | Pfam query aligned with a cytidylate kinase-like family.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |1100px| center | thumb | Top domain results from Pfam.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 400px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; is is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. Also, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful <br /> <br /> [[Image:Possible binding pockets.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling ]]<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> == Laboratory Experiments or ''In vitro'' analysis ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550886 2022-04-26T02:36:47Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |600px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |1300px| center | thumb | Pfam query aligned with a cytidylate kinase-like family.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |1300px| center | thumb | Top domain results from Pfam.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 400px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; is is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. Also, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful <br /> <br /> [[Image:Possible binding pockets.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling ]]<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> == Laboratory Experiments or ''In vitro'' analysis ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550882 2022-04-26T02:33:19Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |600px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |500px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |500px| center | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 400px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; is is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. Also, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful <br /> <br /> [[Image:Possible binding pockets.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling ]]<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> == Laboratory Experiments or ''In vitro'' analysis ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550881 2022-04-26T02:32:49Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |600px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |500px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |500px| center | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 400px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; is is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. Also, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful <br /> <br /> [[Image:Possible binding pockets.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling ]]<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> == Laboratory Experiments or ''In vitro'' analysis ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550880 2022-04-26T02:32:29Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |600px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |500px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |500px| center | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 300px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; is is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. Also, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful <br /> <br /> [[Image:Possible binding pockets.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling ]]<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> == Laboratory Experiments or ''In vitro'' analysis ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550879 2022-04-26T02:31:57Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |600px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |500px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> <br /> [[Image:Pfam search resultsJRAF2022.png |500px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:Pfam domainsJRAF2022.png |500px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 300px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; is is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. Also, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful <br /> <br /> [[Image:Possible binding pockets.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling ]]<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> == Laboratory Experiments or ''In vitro'' analysis ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550768 2022-04-25T15:01:54Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |500px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |500px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> [[Image:3hdt Pfam results.png |500px| center| thumb| Pfam results showing 3HDT is similar to cytidylate kinase-like family proteins.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 300px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; is is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. Also, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful <br /> <br /> [[Image:Possible binding pockets.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling ]]<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> == Laboratory Experiments or ''In vitro'' analysis ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550758 2022-04-25T14:51:38Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLASTp, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |500px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |500px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> [[Image:3hdt Pfam results.png |500px| center| thumb| Pfam results showing 3HDT is similar to cytidylate kinase-like family proteins.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 300px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; is is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. Also, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> <br /> [[Image:Possible binding pockets.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling ]]<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> == Laboratory Experiments or ''In vitro'' analysis ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550757 2022-04-25T14:50:42Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLAST, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |500px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |500px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> [[Image:3hdt Pfam results.png |500px| center| thumb| Pfam results showing 3HDT is similar to cytidylate kinase-like family proteins.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 300px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; is is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. Also, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> <br /> [[Image:Possible binding pockets.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling ]]<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> == Laboratory Experiments or ''In vitro'' analysis ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550756 2022-04-25T14:48:56Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLAST, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show a couple cytidylate kinase-like family proteins almost completely allining with our protein 3HDT.<br /> <br /> [[Image:BLASTp image 1.png |500px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |500px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> [[Image:3hdt Pfam results.png |500px| center| thumb| Pfam results showing 3HDT is similar to cytidylate kinase-like family proteins.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 300px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; is is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. Also, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> <br /> [[Image:Possible binding pockets.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white shperes ]]<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> == Laboratory Experiments or ''In vitro'' analysis ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550755 2022-04-25T14:46:51Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> Beginning with BLAST, we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase.<br /> <br /> [[Image:BLASTp image 1.png |500px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |500px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> [[Image:3hdt Pfam results.png |500px| center| thumb| Pfam results showing 3HDT is similar to cytidylate kinase-like family proteins.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 300px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; is is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. Also, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> <br /> [[Image:Possible binding pockets.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white shperes ]]<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> == Laboratory Experiments or ''In vitro'' analysis ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550754 2022-04-25T14:43:06Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> <br /> [[Image:BLASTp image 1.png |500px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |500px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> [[Image:3hdt Pfam results.png |500px| center| thumb| Pfam results showing 3HDT is similar to cytidylate kinase-like family proteins.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 300px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; is is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. Also, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> <br /> [[Image:Possible binding pockets.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white shperes ]]<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> == Laboratory Experiments or ''In vitro'' analysis ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:3hdt sds page.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester https://proteopedia.org/index.php?title=File:3hdt_sds_page.png&diff=3550753 2022-04-25T14:42:36Z

<p>Autumn Forrester: SDS-PAGE result for the protein 3HDT</p> <hr /> <div>SDS-PAGE result for the protein 3HDT</div>

Autumn Forrester https://proteopedia.org/index.php?title=File:Sds_page_final_pic.png&diff=3550752 2022-04-25T14:41:12Z

<p>Autumn Forrester: uploaded a new version of &quot;Image:Sds page final pic.png&quot;</p> <hr /> <div>SDS-PAGE result for the protein 3HDT</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550749 2022-04-25T14:15:37Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> <br /> [[Image:BLASTp image 1.png |500px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |500px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> [[Image:3hdt Pfam results.png |500px| center| thumb| Pfam results showing 3HDT is similar to cytidylate kinase-like family proteins.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 300px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; is is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. Also, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> <br /> [[Image:Possible binding pockets.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white shperes ]]<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> == Laboratory Experiments or ''In vitro'' analysis ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:Sds page final pic.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester https://proteopedia.org/index.php?title=File:3hdt_Pfam_results.png&diff=3550748 2022-04-25T14:14:03Z

<p>Autumn Forrester: Pfam sequence search results for 3HDT</p> <hr /> <div>Pfam sequence search results for 3HDT</div>

Autumn Forrester https://proteopedia.org/index.php?title=File:BLASTp_image_3.png&diff=3550747 2022-04-25T14:07:18Z

<p>Autumn Forrester: uploaded a new version of &quot;Image:BLASTp image 3.png&quot;: Pfam sequence search results for 3HDT</p> <hr /> <div>Pfam sequence search results for 3HDT</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550746 2022-04-25T13:59:05Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> <br /> [[Image:BLASTp image 1.png |500px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |500px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASTA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> [[Image:BLASTp image 3.png |500px| center| thumb| Pfam results showing cytidylate kinase-like family result.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 300px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; is is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. Also, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> <br /> [[Image:Possible binding pockets.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white shperes ]]<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> == Laboratory Experiments or ''In vitro'' analysis ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:Sds page final pic.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550699 2022-04-24T19:50:07Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> <br /> [[Image:BLASTp image 1.png |500px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |500px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> [[Image:BLASTp image 3.png |500px| center| thumb| Pfam results showing cytidylate kinase-like family result.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:3hdtDALI.png | 300px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; is is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. Also, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> <br /> [[Image:Possible binding pockets.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white shperes ]]<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> == Laboratory Experiments or ''In vitro'' analysis ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:Sds page final pic.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester https://proteopedia.org/index.php?title=File:3hdtDALI.png&diff=3550698 2022-04-24T19:49:40Z

<p>Autumn Forrester: 3hdt aligned with PDB IDs: 7l4a and 1kdo</p> <hr /> <div>3hdt aligned with PDB IDs: 7l4a and 1kdo</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550673 2022-04-24T04:30:31Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> <br /> [[Image:BLASTp image 1.png |500px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |500px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> [[Image:BLASTp image 3.png |500px| center| thumb| Pfam results showing cytidylate kinase-like family result.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:DALI.png | 300px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> This &lt;scene name='90/904996/Binding_pockets/1'&gt;binding pocket&lt;/scene&gt; is is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. Also, this area was where dCMP binded with the highest affinity in PyRx. The amino acids interacting with the substrate within that area are....<br /> <br /> [[Image:Possible binding pockets.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white shperes ]]<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> == Laboratory Experiments or ''In vitro'' analysis ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:Sds page final pic.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550672 2022-04-24T04:26:00Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> <br /> [[Image:BLASTp image 1.png |500px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |500px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> [[Image:BLASTp image 3.png |500px| center| thumb| Pfam results showing cytidylate kinase-like family result.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:DALI.png | 300px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> This &lt;scene name='90/904996/Binding_pockets/1'&gt;potential binding pocket&lt;/scene&gt; is an area where the substrate dCMP may bind on 3HDT. Also, it was an area that dCMP binded to with the most affinity in PyRx. The amino acids interacting with the substrate are....<br /> <br /> [[Image:Possible binding pockets.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white shperes ]]<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> == Laboratory Experiments or ''In vitro'' analysis ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:Sds page final pic.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550671 2022-04-24T04:24:55Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> <br /> [[Image:BLASTp image 1.png |500px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |500px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> [[Image:BLASTp image 3.png |500px| center| thumb| Pfam results showing cytidylate kinase-like family result.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:DALI.png | 300px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> <br /> == Docking==<br /> This &lt;scene name='90/904996/Binding_pockets/1'&gt;potential binding pocket&lt;/scene&gt; is an area where the substrate dCMP may bind on 3HDT. Also, it was an area that dCMP binded to with the most affinity in PyRx. The amino acids interacting with the substrate are....<br /> <br /> [[Image:Possible binding pockets.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white shperes ]]<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> == Laboratory Experiments ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:Sds page final pic.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550670 2022-04-24T04:22:05Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> <br /> [[Image:BLASTp image 1.png |500px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |500px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> [[Image:BLASTp image 3.png |500px| center| thumb| Pfam results showing cytidylate kinase-like family result.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:DALI.png | 300px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> ===='''PANZ'''====<br /> <br /> <br /> == Docking==<br /> This &lt;scene name='90/904996/Binding_pockets/1'&gt;potential binding pocket&lt;/scene&gt; is an area where the substrate dCMP may bind on 3HDT. Also, it was an area that dCMP binded to with the most affinity in PyRx. The amino acids interacting with the substrate are....<br /> <br /> [[Image:Possible binding pockets.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white shperes ]]<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> <br /> <br /> == Laboratory Experiments ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:Sds page final pic.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Also, if we had more time, we would repeat the protein purification process to try to get a higher protein concentration than what we achieved. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550669 2022-04-24T04:16:03Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> <br /> [[Image:BLASTp image 1.png |500px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |500px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> [[Image:BLASTp image 3.png |500px| center| thumb| Pfam results showing cytidylate kinase-like family result.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:DALI.png | 300px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> ===='''PANZ'''====<br /> <br /> <br /> == Docking==<br /> This &lt;scene name='90/904996/Binding_pockets/1'&gt;potential binding pocket&lt;/scene&gt; is an area where the substrate dCMP may bind on 3HDT. Also, it was an area that dCMP binded to with the most affinity in PyRx. The amino acids interacting with the substrate are....<br /> <br /> [[Image:Possible binding pockets.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white shperes ]]<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> <br /> <br /> == Laboratory Experiments ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), there are discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a '''binding metal protein.''' There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:Sds page final pic.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT.  <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester https://proteopedia.org/index.php?title=BASIL2022GV3HDT&diff=3550668 2022-04-24T03:28:51Z

<p>Autumn Forrester: </p> <hr /> <div>==Characterizing Putative Kinase 3HDT==<br /> &lt;StructureSection load='3hdt' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/3'&gt;<br /> Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. <br /> <br /> == Introduction ==<br /> Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein. <br /> <br /> <br /> == ''In silico'' anaylsis ==<br /> We used a variety of ''in silico'' tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP. <br /> <br /> <br /> ===='''BLASTp'''====<br /> <br /> [[Image:BLASTp image 1.png |500px| left | thumb | BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]<br /> <br /> [[Image:BLASTp image 2.png |500px| center| thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]<br /> <br /> <br /> <br /> <br /> ===='''Pfam'''====<br /> We used the FASA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results. <br /> [[Image:BLASTp image 3.png |500px| center| thumb| Pfam results showing cytidylate kinase-like family result.]]<br /> <br /> <br /> ===='''DALI'''====<br /> Two of the top results from our DALI query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of our results from our DALI query consisted of cytidylate kinases. This information helped our decision to choose a function like cytidylate kinase because of similar alignment between the proteins (structure of the protein = function) indicating similarity in function and our protein relates to them.<br /> <br /> <br /> [[Image:DALI.png | 300px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]<br /> <br /> ===='''PANZ'''====<br /> <br /> <br /> == Docking==<br /> This &lt;scene name='90/904996/Binding_pockets/1'&gt;potential binding pocket&lt;/scene&gt; is an area where the substrate dCMP may bind on 3HDT. Also, it was an area that dCMP binded to with the most affinity in PyRx. The amino acids interacting with the substrate are....<br /> <br /> [[Image:Possible binding pockets.png | 400px| center | thumb| Predicted binding pockets for 3HDT represented by the white shperes ]]<br /> <br /> [[Image:DCMP.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink) ]]<br /> [[Image:DCMP reaction.png |250px| center|thumb | Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP]]<br /> <br /> <br /> <br /> <br /> == Laboratory Experiments ==<br /> <br /> ===='''Coupled kinase assay'''====<br /> Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), the results did not turn out the same or even similar to the first round indicating a potential error could have happened. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function. <br /> <br /> <br /> [[Image:Coupled assay.png |400px|left| tumb | representation of the reaction of the coupled assay with 3HDT]]<br /> <br /> [[Image:Coupled assay results.png |500px|center| thumb | Table of data showing all eight coupled kinase assays with 3HDT]]<br /> <br /> Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right). <br /> <br /> <br /> ===='''SDS-PAGE'''====<br /> SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a '''binding metal protein.''' There is also a faint band around ~26kD, indicating our protein of interest was present. <br /> <br /> [[Image:Sds page final pic.png |225px| center | thumb | Results from running an SDS-PAGE with the purified 3HDT]]<br /> <br /> <br /> <br /> == Conclusion/Future Experiments ==<br /> <br /> Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT.  <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 2. National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].<br /> <br /> 3. Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman<br /> Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 4. The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</div>

Autumn Forrester