https://proteopedia.org/api.php?action=feedcontributions&feedformat=atom&user=Michel+Evertsen 2026-09-21T13:05:31Z User contributions MediaWiki 1.43.8 https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3653865 2022-11-01T15:04:58Z

<p>Michel Evertsen: </p> <hr /> <div>=Characterization of the 3r8e Protein, a Novel Glucose Kinase=<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, coupled kinase activity assays, and SDS PAGE analyses were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Previous and subsequent analysis of protein 3r8e validated our initial in silico and in vitro results. Overall, we have strong preliminary evidence that our protein of interest (POI) is a glucose kinase.<br /> == Introduction ==<br /> As apart of a research project under the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) consortium, our group was tasked with characterizing and identifying the function of this protein to provide further insight of the protein's relationship to the bacteria. Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Previous research has shown that there is relationship between our POI and bacteria found in soil. Current research techniques have made the role more apparent and below is the general workflow detailing how we generated our conclusions.<br /> <br /> [[Image:Workflow1.png|500px|]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Structural and sequence alignment analysis with protein 3vov, a hexokinase, is provided below.<br /> <br /> [[Image:3R8EDali180.png|400px|]]<br /> [[Image:3R8EDALIHMM.png|400px|]] <br /> <br /> <br /> From here, we were able to form the conclusion that our POI interacts with glucose based on the alignment with a known hexokinase. To validate that glucose actually binds and interacts with our protein of interest, we conducted a PyRx in silico docking experiment with a total of five hexose substrates. Other substrates tested include fructose, galactose, lactose, and ribose, however, experimental in silico docking results for those substrates were significantly less than glucose. Along with the PyRx docking, we visualized &lt;scene name='90/904995/Glucose_and_atp/1'&gt;ATP and glucose&lt;/scene&gt; within the proposed active site in the PyMol visualization software tool. We also were then able to find which active site amino acid were crucial to binding, which are highlighted &lt;scene name='90/904995/3r8e_amino_acids_updated/3'&gt;here&lt;/scene&gt;. The binding affinity of glucose was -5.1 kcal/mol, which strengthens our idea that glucose is phosphorylated by our protein of interest. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro and because ATP aids in the phosphorylation of glucose, an &lt;scene name='90/904995/3r8ec_w_glc_and_atp/1'&gt;interactive structure&lt;/scene&gt; has been provided to represent interactions of glucose and ATP in the active site.<br /> <br /> <br /> == Experimental Results/Function ==<br /> <br /> Once we felt confident enough to finalize our substrate hypothesis, we began testing in vitro. Beginning with bacterial protein overexpression and affinity chromatography, we were able to purify our POI and begin testing with real substrates. Below are the results of our Uncoupled Kinase Assay, reported in terms of specific activity (mg/mL). Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose. A total of five hexose substrates were tested in vitro, detailed in the table below. Based on these results, we were able to strengthen our initial hypothesis and continue characterization.<br /> <br /> [[Image:SA_1.png|400px|]]<br /> For further validation, we conducted an SDS analysis and provided below is the gel image. Indicated by the black box is our POI, around 34 kDa. Results were not as clear as anticipated, and in future studies, we would need to utilize different chromatography methods to yield higher quality protein concentrations and conduct a pre and post induction to visualize the purity of our protein.<br /> <br /> [[Image:FINAL_gel.png|250px|]]<br /> <br /> == Project Implications ==<br /> The goal of this project is to explore the techniques it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, including bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE. These techniques can be used to help characterize further putative kinases discovered in the future that do not have a defined function. This project is of importance because proteins are biomolecules responsible for as organisms' survival and understanding their unique function is essential for advances in modern medicine, scientific research, and agriculture.<br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> After various experiments and discussion, we have concluded that the novel protein 3R8E is a &lt;scene name='90/904995/Mesh_backbone_with_atp_glucose/3'&gt;Glucose Kinase&lt;/scene&gt;. By using an in-vitro assay, we obtained results comparing the phosphorylation rates of five sugars in the presence of our putative kinase. From the assay results, we were able to calculate an average specific activity for all experimental sugars, and by comparing these numbers we can clearly see that glucose is being phosphorylated in the presence of protein 3R8E. To get to this conclusion, we ran triplicates of the experiment for each sugar, and then followed a calculation procedure to get specific activity numbers to quantify how active our protein is with a given substrate. By doing this, we were able to validate and further support our hypothesis, which now can allow others to replicate or continue our research. Glucose provided us with a specific activity value of 0.214 +/- 0.893 and 1.223 +/- 2.575 for prep one and two respectively. <br /> <br /> Future Direction:<br /> <br /> After validating our results, we now look to take our findings to a micropublication website for undergraduate research. By doing this, not only will our work be forward facing and available to the science community, but it also allows for collaboration and further questions to be asked. After completing the micropublication, we look to continue to develop research strategies for putative kinases, as the PDB has thousands of proteins with unsolved functions. We will do this by combining machine learning, data science, and lab work to allow undergraduate students and scientist to effectively research and study putative kinase structures and functions. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3647024 2022-10-19T19:34:44Z

<p>Michel Evertsen: </p> <hr /> <div>=Characterization of the 3r8e Protein, a Novel Glucose Kinase=<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, coupled kinase activity assays, and SDS PAGE analyses were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Previous and subsequent analysis of protein 3r8e validated our initial in silico and in vitro results. Overall, we have strong preliminary evidence that our protein of interest (POI) is a glucose kinase.<br /> == Introduction ==<br /> As apart of a research project under the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) consortium, our group was tasked with characterizing and identifying the function of this protein to provide further insight of the protein's relationship to the bacteria. Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Previous research has shown that there is relationship between our POI and bacteria found in soil. Current research techniques have made the role more apparent and below is the general workflow detailing how we generated our conclusions.<br /> <br /> [[Image:Workflow1.png|500px|]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Structural and sequence alignment analysis with protein 3vov, a hexokinase, is provided below.<br /> <br /> [[Image:3R8EDali180.png|400px|]]<br /> [[Image:3R8EDALIHMM.png|400px|]] <br /> <br /> <br /> From here, we were able to form the conclusion that our POI interacts with glucose based on the alignment with a known hexokinase. To validate that glucose actually binds and interacts with our protein of interest, we conducted a PyRx in silico docking experiment with a total of five hexose substrates. Other substrates tested include fructose, galactose, lactose, and ribose, however, experimental in silico docking results for those substrates were significantly less than glucose. Along with the PyRx docking, we visualized &lt;scene name='90/904995/Glucose_and_atp/1'&gt;ATP and glucose&lt;/scene&gt; within the proposed active site in the PyMol visualization software tool. We also were then able to find which active site amino acid were crucial to binding, which are highlighted &lt;scene name='90/904995/3r8e_amino_acids_nolig/1'&gt;here&lt;/scene&gt;. The binding affinity of glucose was -5.1 kcal/mol, which strengthens our idea that glucose is phosphorylated by our protein of interest. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro and because ATP aids in the phosphorylation of glucose, an &lt;scene name='90/904995/3r8ec_w_glc_and_atp/1'&gt;interactive structure&lt;/scene&gt; has been provided to represent interactions of glucose and ATP in the active site.<br /> <br /> <br /> == Experimental Results/Function ==<br /> <br /> Once we felt confident enough to finalize our substrate hypothesis, we began testing in vitro. Beginning with bacterial protein overexpression and affinity chromatography, we were able to purify our POI and begin testing with real substrates. Below are the results of our Uncoupled Kinase Assay, reported in terms of specific activity (mg/mL). Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose. A total of five hexose substrates were tested in vitro, detailed in the table below. Based on these results, we were able to strengthen our initial hypothesis and continue characterization.<br /> <br /> [[Image:Specific_activity_table.png|400px|]]<br /> For further validation, we conducted an SDS analysis and provided below is the gel image. Indicated by the black box is our POI, around 34 kDa. Results were not as clear as anticipated, and in future studies, we would need to utilize different chromatography methods to yield higher quality protein concentrations and conduct a pre and post induction to visualize the purity of our protein.<br /> <br /> [[Image:FINAL_gel.png|250px|]]<br /> <br /> == Project Implications ==<br /> The goal of this project is to explore the techniques it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, including bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE. These techniques can be used to help characterize further putative kinases discovered in the future that do not have a defined function. This project is of importance because proteins are biomolecules responsible for as organisms' survival and understanding their unique function is essential for advances in modern medicine, scientific research, and agriculture.<br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> After various experiments and discussion, we have concluded that the novel protein 3R8E is a &lt;scene name='90/904995/Mesh_backbone_with_atp_glucose/3'&gt;Glucose Kinase&lt;/scene&gt;. By using an in-vitro assay, we obtained results comparing the phosphorylation rates of five sugars in the presence of our putative kinase. From the assay results, we were able to calculate an average specific activity for all experimental sugars, and by comparing these numbers we can clearly see that glucose is being phosphorylated in the presence of protein 3R8E. To get to this conclusion, we ran triplicates of the experiment for each sugar, and then followed a calculation procedure to get specific activity numbers to quantify how active our protein is with a given substrate. By doing this, we were able to validate and further support our hypothesis, which now can allow others to replicate or continue our research. Glucose provided us with a specific activity value of 0.214 +/- 0.893 and 1.223 +/- 2.575 for prep one and two respectively. <br /> <br /> Future Direction:<br /> <br /> After validating our results, we now look to take our findings to a micropublication website for undergraduate research. By doing this, not only will our work be forward facing and available to the science community, but it also allows for collaboration and further questions to be asked. After completing the micropublication, we look to continue to develop research strategies for putative kinases, as the PDB has thousands of proteins with unsolved functions. We will do this by combining machine learning, data science, and lab work to allow undergraduate students and scientist to effectively research and study putative kinase structures and functions. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3646493 2022-10-19T19:22:47Z

<p>Michel Evertsen: </p> <hr /> <div>=Characterization of the 3r8e Protein, a Novel Glucose Kinase=<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, coupled kinase activity assays, and SDS PAGE analyses were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Previous and subsequent analysis of protein 3r8e validated our initial in silico and in vitro results. Overall, we have strong preliminary evidence that our protein of interest (POI) is a glucose kinase.<br /> == Introduction ==<br /> As apart of a research project under the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) consortium, our group was tasked with characterizing and identifying the function of this protein to provide further insight of the protein's relationship to the bacteria. Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Previous research has shown that there is relationship between our POI and bacteria found in soil. Current research techniques have made the role more apparent and below is the general workflow detailing how we generated our conclusions.<br /> <br /> [[Image:Workflow1.png|500px|]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Structural and sequence alignment analysis with protein 3vov, a hexokinase, is provided below.<br /> <br /> [[Image:3R8EDali180.png|400px|]]<br /> [[Image:3R8EDALIHMM.png|400px|]] <br /> <br /> <br /> From here, we were able to form the conclusion that our POI interacts with glucose based on the alignment with a known hexokinase. To validate that glucose actually binds and interacts with our protein of interest, we conducted a PyRx in silico docking experiment with a total of five hexose substrates. Other substrates tested include fructose, galactose, lactose, and ribose, however, experimental in silico docking results for those substrates were significantly less than glucose. Along with the PyRx docking, we visualized ATP and &lt;scene name='90/904995/Mesh_backbone_with_atp_glucose/3'&gt;glucose&lt;/scene&gt; within the proposed active site in the PyMol visualization software tool. We also were then able to find which active site amino acid were crucial to binding, which are highlighted &lt;scene name='90/904995/3r8e_amino_acids_nolig/1'&gt;here&lt;/scene&gt;. The binding affinity of glucose was -5.1 kcal/mol, which strengthens our idea that glucose is phosphorylated by our protein of interest. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro and because ATP aids in the phosphorylation of glucose, an &lt;scene name='90/904995/3r8ec_w_glc_and_atp/1'&gt;interactive structure&lt;/scene&gt; has been provided to represent interactions of glucose and ATP in the active site.<br /> <br /> <br /> == Experimental Results/Function ==<br /> <br /> Once we felt confident enough to finalize our substrate hypothesis, we began testing in vitro. Beginning with bacterial protein overexpression and affinity chromatography, we were able to purify our POI and begin testing with real substrates. Below are the results of our Uncoupled Kinase Assay, reported in terms of specific activity (mg/mL). Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose. A total of five hexose substrates were tested in vitro, detailed in the table below. Based on these results, we were able to strengthen our initial hypothesis and continue characterization.<br /> <br /> [[Image:Specific_activity_table.png|400px|]]<br /> For further validation, we conducted an SDS analysis and provided below is the gel image. Indicated by the black box is our POI, around 34 kDa. Results were not as clear as anticipated, and in future studies, we would need to utilize different chromatography methods to yield higher quality protein concentrations and conduct a pre and post induction to visualize the purity of our protein.<br /> <br /> [[Image:FINAL_gel.png|250px|]]<br /> <br /> == Project Implications ==<br /> The goal of this project is to explore the techniques it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, including bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE. These techniques can be used to help characterize further putative kinases discovered in the future that do not have a defined function. This project is of importance because proteins are biomolecules responsible for as organisms' survival and understanding their unique function is essential for advances in modern medicine, scientific research, and agriculture.<br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> After various experiments and discussion, we have concluded that the novel protein 3R8E is a &lt;scene name='90/904995/Mesh_backbone_with_atp_glucose/3'&gt;Glucose Kinase&lt;/scene&gt;. By using an in-vitro assay, we obtained results comparing the phosphorylation rates of five sugars in the presence of our putative kinase. From the assay results, we were able to calculate an average specific activity for all experimental sugars, and by comparing these numbers we can clearly see that glucose is being phosphorylated in the presence of protein 3R8E. To get to this conclusion, we ran triplicates of the experiment for each sugar, and then followed a calculation procedure to get specific activity numbers to quantify how active our protein is with a given substrate. By doing this, we were able to validate and further support our hypothesis, which now can allow others to replicate or continue our research. Glucose provided us with a specific activity value of 0.214 +/- 0.893 and 1.223 +/- 2.575 for prep one and two respectively. <br /> <br /> Future Direction:<br /> <br /> After validating our results, we now look to take our findings to a micropublication website for undergraduate research. By doing this, not only will our work be forward facing and available to the science community, but it also allows for collaboration and further questions to be asked. After completing the micropublication, we look to continue to develop research strategies for putative kinases, as the PDB has thousands of proteins with unsolved functions. We will do this by combining machine learning, data science, and lab work to allow undergraduate students and scientist to effectively research and study putative kinase structures and functions. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3646255 2022-10-19T19:15:34Z

<p>Michel Evertsen: </p> <hr /> <div>=Characterization of the 3r8e Protein, a Novel Glucose Kinase=<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, coupled kinase activity assays, and SDS PAGE analyses were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Previous and subsequent analysis of protein 3r8e validated our initial in silico and in vitro results. Overall, we have strong preliminary evidence that our protein of interest (POI) is a glucose kinase.<br /> == Introduction ==<br /> As apart of a research project under the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) consortium, our group was tasked with characterizing and identifying the function of this protein to provide further insight of the protein's relationship to the bacteria. Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Previous research has shown that there is relationship between our POI and bacteria found in soil. Current research techniques have made the role more apparent and below is the general workflow detailing how we generated our conclusions.<br /> <br /> [[Image:Workflow1.png|500px|]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Structural and sequence alignment analysis with protein 3vov, a hexokinase, is provided below.<br /> <br /> [[Image:3R8EDali180.png|400px|]]<br /> [[Image:3R8EDALIHMM.png|400px|]] <br /> <br /> <br /> From here, we were able to form the conclusion that our POI interacts with glucose based on the alignment with a known hexokinase. To validate that glucose actually binds and interacts with our protein of interest, we conducted a PyRx in silico docking experiment with a total of five hexose substrates. Other substrates tested include fructose, galactose, lactose, and ribose, however, experimental in silico docking results for those substrates were significantly less than glucose. Along with the PyRx docking, we visualized ATP and &lt;scene name='90/904995/Mesh_backbone_with_atp_glucose/3'&gt;glucose&lt;/scene&gt; within the proposed active site in the PyMol visualization software tool. We also were then able to find which active site amino acid were crucial to binding, which are highlighted &lt;scene name='90/904995/3r8e_amino_acids_updated/2'&gt;here&lt;/scene&gt;. The binding affinity of glucose was -5.1 kcal/mol, which strengthens our idea that glucose is phosphorylated by our protein of interest. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro and because ATP aids in the phosphorylation of glucose, an &lt;scene name='90/904995/3r8ec_w_glc_and_atp/1'&gt;interactive structure&lt;/scene&gt; has been provided to represent interactions of glucose and ATP in the active site.<br /> <br /> <br /> == Experimental Results/Function ==<br /> <br /> Once we felt confident enough to finalize our substrate hypothesis, we began testing in vitro. Beginning with bacterial protein overexpression and affinity chromatography, we were able to purify our POI and begin testing with real substrates. Below are the results of our Uncoupled Kinase Assay, reported in terms of specific activity (mg/mL). Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose. A total of five hexose substrates were tested in vitro, detailed in the table below. Based on these results, we were able to strengthen our initial hypothesis and continue characterization.<br /> <br /> [[Image:Specific_activity_table.png|400px|]]<br /> For further validation, we conducted an SDS analysis and provided below is the gel image. Indicated by the black box is our POI, around 34 kDa. Results were not as clear as anticipated, and in future studies, we would need to utilize different chromatography methods to yield higher quality protein concentrations and conduct a pre and post induction to visualize the purity of our protein.<br /> <br /> [[Image:FINAL_gel.png|250px|]]<br /> <br /> == Project Implications ==<br /> The goal of this project is to explore the techniques it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, including bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE. These techniques can be used to help characterize further putative kinases discovered in the future that do not have a defined function. This project is of importance because proteins are biomolecules responsible for as organisms' survival and understanding their unique function is essential for advances in modern medicine, scientific research, and agriculture.<br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> After various experiments and discussion, we have concluded that the novel protein 3R8E is a &lt;scene name='90/904995/Mesh_backbone_with_atp_glucose/3'&gt;Glucose Kinase&lt;/scene&gt;. By using an in-vitro assay, we obtained results comparing the phosphorylation rates of five sugars in the presence of our putative kinase. From the assay results, we were able to calculate an average specific activity for all experimental sugars, and by comparing these numbers we can clearly see that glucose is being phosphorylated in the presence of protein 3R8E. To get to this conclusion, we ran triplicates of the experiment for each sugar, and then followed a calculation procedure to get specific activity numbers to quantify how active our protein is with a given substrate. By doing this, we were able to validate and further support our hypothesis, which now can allow others to replicate or continue our research. Glucose provided us with a specific activity value of 0.214 +/- 0.893 and 1.223 +/- 2.575 for prep one and two respectively. <br /> <br /> Future Direction:<br /> <br /> After validating our results, we now look to take our findings to a micropublication website for undergraduate research. By doing this, not only will our work be forward facing and available to the science community, but it also allows for collaboration and further questions to be asked. After completing the micropublication, we look to continue to develop research strategies for putative kinases, as the PDB has thousands of proteins with unsolved functions. We will do this by combining machine learning, data science, and lab work to allow undergraduate students and scientist to effectively research and study putative kinase structures and functions. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3646254 2022-10-19T19:06:48Z

<p>Michel Evertsen: </p> <hr /> <div>=Characterization of the 3r8e Protein, a Novel Glucose Kinase=<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, coupled kinase activity assays, and SDS PAGE analyses were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Previous and subsequent analysis of protein 3r8e validated our initial in silico and in vitro results. Overall, we have strong preliminary evidence that our protein of interest (POI) is a glucose kinase.<br /> == Introduction ==<br /> As apart of a research project under the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) consortium, our group was tasked with characterizing and identifying the function of this protein to provide further insight of the protein's relationship to the bacteria. Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Previous research has shown that there is relationship between our POI and bacteria found in soil. Current research techniques have made the role more apparent and below is the general workflow detailing how we generated our conclusions.<br /> <br /> [[Image:Workflow1.png|500px|]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Structural and sequence alignment analysis with protein 3vov, a hexokinase, is provided below.<br /> <br /> [[Image:3R8EDali180.png|400px|]]<br /> [[Image:3R8EDALIHMM.png|400px|]] <br /> <br /> <br /> From here, we were able to form the conclusion that our POI interacts with glucose based on the alignment with a known hexokinase. To validate that glucose actually binds and interacts with our protein of interest, we conducted a PyRx in silico docking experiment with a total of five hexose substrates. Other substrates tested include fructose, galactose, lactose, and ribose, however, experimental in silico docking results for those substrates were significantly less than glucose. Along with the PyRx docking, we visualized ATP and &lt;scene name='90/904995/Mesh_backbone_with_atp_glucose/3'&gt;glucose&lt;/scene&gt; within the proposed active site in the PyMol visualization software tool. We also were then able to find which active site amino acid were crucial to binding, which are highlighted &lt;scene name='90/904995/3r8e_amino_acids/2'&gt;here&lt;/scene&gt;.&lt;scene name='90/904995/3r8e_amino_acids_updated/1'&gt;here&lt;/scene&gt;. The binding affinity of glucose was -5.1 kcal/mol, which strengthens our idea that glucose is phosphorylated by our protein of interest. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro and because ATP aids in the phosphorylation of glucose, an &lt;scene name='90/904995/3r8ec_w_glc_and_atp/1'&gt;interactive structure&lt;/scene&gt; has been provided to represent interactions of glucose and ATP in the active site.<br /> <br /> <br /> == Experimental Results/Function ==<br /> <br /> Once we felt confident enough to finalize our substrate hypothesis, we began testing in vitro. Beginning with bacterial protein overexpression and affinity chromatography, we were able to purify our POI and begin testing with real substrates. Below are the results of our Uncoupled Kinase Assay, reported in terms of specific activity (mg/mL). Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose. A total of five hexose substrates were tested in vitro, detailed in the table below. Based on these results, we were able to strengthen our initial hypothesis and continue characterization.<br /> <br /> [[Image:Specific_activity_table.png|400px|]]<br /> For further validation, we conducted an SDS analysis and provided below is the gel image. Indicated by the black box is our POI, around 34 kDa. Results were not as clear as anticipated, and in future studies, we would need to utilize different chromatography methods to yield higher quality protein concentrations and conduct a pre and post induction to visualize the purity of our protein.<br /> <br /> [[Image:FINAL_gel.png|250px|]]<br /> <br /> == Project Implications ==<br /> The goal of this project is to explore the techniques it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, including bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE. These techniques can be used to help characterize further putative kinases discovered in the future that do not have a defined function. This project is of importance because proteins are biomolecules responsible for as organisms' survival and understanding their unique function is essential for advances in modern medicine, scientific research, and agriculture.<br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> After various experiments and discussion, we have concluded that the novel protein 3R8E is a &lt;scene name='90/904995/Mesh_backbone_with_atp_glucose/3'&gt;Glucose Kinase&lt;/scene&gt;. By using an in-vitro assay, we obtained results comparing the phosphorylation rates of five sugars in the presence of our putative kinase. From the assay results, we were able to calculate an average specific activity for all experimental sugars, and by comparing these numbers we can clearly see that glucose is being phosphorylated in the presence of protein 3R8E. To get to this conclusion, we ran triplicates of the experiment for each sugar, and then followed a calculation procedure to get specific activity numbers to quantify how active our protein is with a given substrate. By doing this, we were able to validate and further support our hypothesis, which now can allow others to replicate or continue our research. Glucose provided us with a specific activity value of 0.214 +/- 0.893 and 1.223 +/- 2.575 for prep one and two respectively. <br /> <br /> Future Direction:<br /> <br /> After validating our results, we now look to take our findings to a micropublication website for undergraduate research. By doing this, not only will our work be forward facing and available to the science community, but it also allows for collaboration and further questions to be asked. After completing the micropublication, we look to continue to develop research strategies for putative kinases, as the PDB has thousands of proteins with unsolved functions. We will do this by combining machine learning, data science, and lab work to allow undergraduate students and scientist to effectively research and study putative kinase structures and functions. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3643995 2022-10-10T03:06:54Z

<p>Michel Evertsen: </p> <hr /> <div>=Characterization of the 3r8e Protein, a Novel Glucose Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, coupled kinase activity assays, and SDS PAGE analyses were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Previous and subsequent analysis of protein 3r8e validated our initial in silico and in vitro results. Overall, we have strong preliminary evidence that the our protein of interest (POI) is a glucose kinase.<br /> == Introduction ==<br /> As apart of a research project under the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) consortium, our group was tasked with characterizing and identifying the function of this protein to provide further insight of the protein's relationship to the bacteria. Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Previous research has shown that there is relationship between our POI and bacteria found in soil. Current research techniques have made the role more apparent and below is the general workflow detailing how we generated our conclusions.<br /> <br /> [[Image:Workflow1.png|500px|]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Structural and sequence alignment analysis with protein 3vov, a hexokinase, is provided below.<br /> <br /> [[Image:3R8EDali180.png|400px|]]<br /> [[Image:3R8EDALIHMM.png|400px|]] <br /> <br /> <br /> From here, we were able to form the conclusion that our POI interacts with glucose based on the alignment with a known hexokinase. To validate that glucose actually binds and interacts with our protein of interest, we conducted a PyRx in silico docking experiment with a total of five hexose substrates. Other substrates tested include fructose, galactose, lactose, and ribose, however, experimental in silico docking results for those substrates were significantly less than glucose. Along with the PyRx docking, we visualized ATP and &lt;scene name='90/904995/Mesh_backbone_with_atp_glucose/3'&gt;glucose&lt;/scene&gt; within the proposed active site in the PyMol visualization software tool. We also were then able to find which active site amino acid were crucial to binding, which are highlighted &lt;scene name='90/904995/3r8e_amino_acids/2'&gt;here&lt;/scene&gt;. The binding affinity of glucose was -5.1 kcal/mol, which strengthens our idea that glucose is phosphorylated by our protein of interest. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro and because ATP aids in the phosphorylation of glucose, an &lt;scene name='90/904995/3r8ec_w_glc_and_atp/1'&gt;interactive structure&lt;/scene&gt; has been provided to represent interactions of glucose and ATP in the active site.<br /> <br /> <br /> == Experimental Results/Function ==<br /> <br /> Once we felt confident enough to finalize our substrate hypothesis, we began testing in vitro. Beginning with bacterial protein overexpression and affinity chromatography, we were able to purify our POI and begin testing with real substrates. Below are the results of our Uncoupled Kinase Assay, reported in terms of specific activity (mg/mL). Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose. A total of five hexose substrates were tested in vitro, detailed in the table below. Based on these results, we were able to strengthen our initial hypothesis and continue characterization.<br /> <br /> [[Image:Specific_activity_table.png|400px|]]<br /> For further validation, we conducted an SDS analysis and provided below is the gel image. Indicated by the black box is our POI, around 34 kDa. Results were not as clear as anticipated, and in future studies, we would need to utilize different chromatography methods to yield higher quality protein concentrations and conduct a pre and post induction to visualize the purity of our protein.<br /> <br /> [[Image:FINAL_gel.png|250px|]]<br /> <br /> == Project Implications ==<br /> The goal of this project is to explore the techniques it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, including bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE. These techniques can be used to help characterize further putative kinases discovered in the future that do not have a defined function. This project is of importance because proteins are biomolecules responsible for as organisms' survival and understanding their unique function is essential for advances in modern medicine, scientific research, and agriculture.<br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> After various experiments and discussion, we have concluded that the novel protein 3R8E is a &lt;scene name='90/904995/Mesh_backbone_with_atp_glucose/3'&gt;Glucose Kinase&lt;/scene&gt;. By using an in-vitro assay, we obtained results comparing the phosphorylation rates of five sugars in the presence of our putative kinase. From the assay results, we were able to calculate an average specific activity for all experimental sugars, and by comparing these numbers we can clearly see that glucose is being phosphorylated in the presence of protein 3R8E. To get to this conclusion, we ran triplicates of the experiment for each sugar, and then followed a calculation procedure to get specific activity numbers to quantify how active our protein is with a given substrate. By doing this, we were able to validate and further support our hypothesis, which now can allow others to replicate or continue our research. Glucose provided us with a specific activity value of 0.214 +/- 0.893 and 1.223 +/- 2.575 for prep one and two respectively. <br /> <br /> Future Direction:<br /> <br /> After validating our results, we now look to take our findings to a micropublication website for undergraduate research. By doing this, not only will our work be forward facing and available to the science community, but it also allows for collaboration and further questions to be asked. After completing the micropublication, we look to continue to develop research strategies for putative kinases, as the PDB has thousands of proteins with unsolved functions. We will do this by combining machine learning, data science, and lab work to allow undergraduate students and scientist to effectively research and study putative kinase structures and functions. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3641369 2022-10-05T18:34:12Z

<p>Michel Evertsen: </p> <hr /> <div>&lt;scene name='90/904995/3r8ec_w_glc_and_atp/2'&gt;Text To Be Displayed&lt;/scene&gt;==Characterization of the 3r8e Protein, a Novel Glucose Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, coupled kinase activity assays, and SDS PAGE analyses were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Previous and subsequent analysis of protein 3r8e validated our initial in silico and in vitro results. Overall, we have strong preliminary evidence that the our protein of interest (POI) is a glucose kinase.<br /> == Introduction ==<br /> As apart of a research project under the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) consortium, our group was tasked with characterizing and identifying the function of this protein to provide further insight of the protein's relationship to the bacteria. Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Previous research has shown that there is relationship between our POI and bacteria found in soil. Current research techniques have made the role more apparent and below is the general workflow detailing how we generated our conclusions.<br /> <br /> [[Image:Workflow1.png|500px|]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Structural and sequence alignment analysis with protein 3vov, a hexokinase, is provided below.<br /> <br /> [[Image:3R8EDali180.png|400px|]]<br /> [[Image:3R8EDALIHMM.png|400px|]] <br /> <br /> <br /> From here, we were able to form the conclusion that our POI interacts with glucose based on the alignment with a known hexokinase. To validate that glucose actually binds and interacts with our protein of interest, we conducted a PyRx in silico docking experiment with a total of five hexose substrates. Other substrates tested include fructose, galactose, lactose, and ribose, however, experimental in silico docking results for those substrates were significantly less than glucose. Along with the PyRx docking, we visualized ATP and &lt;scene name='90/904995/Mesh_backbone_with_atp_glucose/3'&gt;glucose&lt;/scene&gt; within the proposed active site in the PyMol visualization software tool. We also were then able to find which active site amino acid were crucial to binding, which are highlighted &lt;scene name='90/904995/3r8e_amino_acids/2'&gt;here&lt;/scene&gt;. The binding affinity of glucose was -5.1 kcal/mol, which strengthens our idea that glucose is phosphorylated by our protein of interest. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro and because ATP aids in the phosphorylation of glucose, an &lt;scene name='90/904995/3r8ec_w_glc_and_atp/1'&gt;interactive structure&lt;/scene&gt; has been provided to represent interactions of glucose and ATP in the active site.<br /> <br /> <br /> == Experimental Results/Function ==<br /> <br /> Once we felt confident enough to finalize our substrate hypothesis, we began testing in vitro. Beginning with bacterial protein overexpression and affinity chromatography, we were able to purify our POI and begin testing with real substrates. Below are the results of our Uncoupled Kinase Assay, reported in terms of specific activity (mg/mL). Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose. A total of five hexose substrates were tested in vitro, detailed in the table below. Based on these results, we were able to strengthen our initial hypothesis and continue characterization.<br /> <br /> [[Image:Specific_activity_table.png|400px|]]<br /> For further validation, we conducted an SDS analysis and provided below is the gel image. Indicated by the black box is our POI, around 34 kDa. Results were not as clear as anticipated, and in future studies, we would need to utilize different chromatography methods to yield higher quality protein concentrations and conduct a pre and post induction to visualize the purity of our protein.<br /> <br /> [[Image:FINAL_gel.png|250px|]]<br /> <br /> == Project Implications ==<br /> The goal of this project is to explore the techniques it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, including bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE. These techniques can be used to help characterize further putative kinases discovered in the future that do not have a defined function. This project is of importance because proteins are biomolecules responsible for as organisms' survival and understanding their unique function is essential for advances in modern medicine, scientific research, and agriculture.<br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> After various experiments and discussion, we have concluded that the novel protein 3R8E is a &lt;scene name='90/904995/Mesh_backbone_with_atp_glucose/3'&gt;Glucose Kinase&lt;/scene&gt;. By using an in-vitro assay, we obtained results comparing the phosphorylation rates of five sugars in the presence of our putative kinase. From the assay results, we were able to calculate an average specific activity for all experimental sugars, and by comparing these numbers we can clearly see that glucose is being phosphorylated in the presence of protein 3R8E. To get to this conclusion, we ran triplicates of the experiment for each sugar, and then followed a calculation procedure to get specific activity numbers to quantify how active our protein is with a given substrate. By doing this, we were able to validate and further support our hypothesis, which now can allow others to replicate or continue our research. Glucose provided us with a specific activity value of 0.214 +/- 0.893 and 1.223 +/- 2.575 for prep one and two respectively. <br /> <br /> Future Direction:<br /> <br /> After validating our results, we now look to take our findings to a micropublication website for undergraduate research. By doing this, not only will our work be forward facing and available to the science community, but it also allows for collaboration and further questions to be asked. After completing the micropublication, we look to continue to develop research strategies for putative kinases, as the PDB has thousands of proteins with unsolved functions. We will do this by combining machine learning, data science, and lab work to allow undergraduate students and scientist to effectively research and study putative kinase structures and functions. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3641368 2022-10-05T18:32:18Z

<p>Michel Evertsen: </p> <hr /> <div>&lt;scene name='90/904995/3r8ec_w_glc_and_atp/2'&gt;Text To Be Displayed&lt;/scene&gt;==Characterization of the 3r8e Protein, a Novel Glucose Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, coupled kinase activity assays, and SDS PAGE analyses were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Previous and subsequent analysis of protein 3r8e validated our initial in silico and in vitro results. Overall, we have strong preliminary evidence that the our protein of interest (POI) is a glucose kinase.<br /> == Introduction ==<br /> As apart of a research project under the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) consortium, our group was tasked with characterizing and identifying the function of this protein to provide further insight of the protein's relationship to the bacteria. Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Previous research has shown that there is relationship between our POI and bacteria found in soil. Current research techniques have made the role more apparent and below is the general workflow detailing how we generated our conclusions.<br /> <br /> [[Image:Workflow1.png|500px|]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Structural and sequence alignment analysis with protein 3vov, a hexokinase, is provided below.<br /> <br /> [[Image:3R8EDali180.png|400px|]]<br /> [[Image:3R8EDALIHMM.png|400px|]] <br /> <br /> <br /> From here, we were able to form the conclusion that our POI interacts with glucose based on the alignment with a known hexokinase. To validate that glucose actually binds and interacts with our protein of interest, we conducted a PyRx in silico docking experiment with a total of five hexose substrates. Other substrates tested include fructose, galactose, lactose, and ribose, however, experimental in silico docking results for those substrates were significantly less than glucose. Along with the PyRx docking, we visualized ATP and glucose (Orange) within the proposed active site in the PyMol visualization below. We also were then able to find which active site amino acid were crucial to binding, which are highlighted &lt;scene name='90/904995/3r8e_amino_acids/2'&gt;here&lt;/scene&gt;. The binding affinity of glucose was -5.1 kcal/mol, which strengthens our idea that glucose is phosphorylated by our protein of interest. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro and because ATP aids in the phosphorylation of glucose, an &lt;scene name='90/904995/3r8ec_w_glc_and_atp/1'&gt;interactive structure&lt;/scene&gt; has been provided to represent interactions of glucose and ATP in the active site.<br /> <br /> <br /> == Experimental Results/Function ==<br /> <br /> Once we felt confident enough to finalize our substrate hypothesis, we began testing in vitro. Beginning with bacterial protein overexpression and affinity chromatography, we were able to purify our POI and begin testing with real substrates. Below are the results of our Uncoupled Kinase Assay, reported in terms of specific activity (mg/mL). Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose. A total of five hexose substrates were tested in vitro, detailed in the table below. Based on these results, we were able to strengthen our initial hypothesis and continue characterization.<br /> <br /> [[Image:Specific_activity_table.png|400px|]]<br /> For further validation, we conducted an SDS analysis and provided below is the gel image. Indicated by the black box is our POI, around 34 kDa. Results were not as clear as anticipated, and in future studies, we would need to utilize different chromatography methods to yield higher quality protein concentrations and conduct a pre and post induction to visualize the purity of our protein.<br /> <br /> [[Image:FINAL_gel.png|250px|]]<br /> <br /> == Project Implications ==<br /> The goal of this project is to explore the techniques it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, including bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE. These techniques can be used to help characterize further putative kinases discovered in the future that do not have a defined function. This project is of importance because proteins are biomolecules responsible for as organisms' survival and understanding their unique function is essential for advances in modern medicine, scientific research, and agriculture.<br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> After various experiments and discussion, we have concluded that the novel protein 3R8E is a &lt;scene name='90/904995/Mesh_backbone_with_atp_glucose/3'&gt;Glucose Kinase&lt;/scene&gt;. By using an in-vitro assay, we obtained results comparing the phosphorylation rates of five sugars in the presence of our putative kinase. From the assay results, we were able to calculate an average specific activity for all experimental sugars, and by comparing these numbers we can clearly see that glucose is being phosphorylated in the presence of protein 3R8E. To get to this conclusion, we ran triplicates of the experiment for each sugar, and then followed a calculation procedure to get specific activity numbers to quantify how active our protein is with a given substrate. By doing this, we were able to validate and further support our hypothesis, which now can allow others to replicate or continue our research. Glucose provided us with a specific activity value of 0.214 +/- 0.893 and 1.223 +/- 2.575 for prep one and two respectively. <br /> <br /> Future Direction:<br /> <br /> After validating our results, we now look to take our findings to a micropublication website for undergraduate research. By doing this, not only will our work be forward facing and available to the science community, but it also allows for collaboration and further questions to be asked. After completing the micropublication, we look to continue to develop research strategies for putative kinases, as the PDB has thousands of proteins with unsolved functions. We will do this by combining machine learning, data science, and lab work to allow undergraduate students and scientist to effectively research and study putative kinase structures and functions. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3641367 2022-10-05T18:29:43Z

<p>Michel Evertsen: </p> <hr /> <div>&lt;scene name='90/904995/3r8ec_w_glc_and_atp/2'&gt;Text To Be Displayed&lt;/scene&gt;==Characterization of the 3r8e Protein, a Novel Glucose Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, coupled kinase activity assays, and SDS PAGE analyses were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Previous and subsequent analysis of protein 3r8e validated our initial in silico and in vitro results. Overall, we have strong preliminary evidence that the our protein of interest (POI) is a glucose kinase.<br /> == Introduction ==<br /> As apart of a research project under the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) consortium, our group was tasked with characterizing and identifying the function of this protein to provide further insight of the protein's relationship to the bacteria. Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Previous research has shown that there is relationship between our POI and bacteria found in soil. Current research techniques have made the role more apparent and below is the general workflow detailing how we generated our conclusions.<br /> <br /> [[Image:Workflow1.png|500px|]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Structural and sequence alignment analysis with protein 3vov, a hexokinase, is provided below.<br /> <br /> [[Image:3R8EDali180.png|400px|]]<br /> [[Image:3R8EDALIHMM.png|400px|]] <br /> <br /> <br /> From here, we were able to form the conclusion that our POI interacts with glucose based on the alignment with a known hexokinase. To validate that glucose actually binds and interacts with our protein of interest, we conducted a PyRx in silico docking experiment with a total of five hexose substrates. Other substrates tested include fructose, galactose, lactose, and ribose, however, experimental in silico docking results for those substrates were significantly less than glucose. Along with the PyRx docking, we visualized ATP and glucose (Orange) within the proposed active site in the PyMol visualization below. The binding affinity of glucose was -5.1 kcal/mol, which strengthens our idea that glucose is phosphorylated by our protein of interest. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro and because ATP aids in the phosphorylation of glucose, an &lt;scene name='90/904995/3r8ec_w_glc_and_atp/1'&gt;interactive structure&lt;/scene&gt; has been provided to represent interactions of glucose and ATP in the active site.<br /> <br /> &lt;scene name='90/904995/3r8e_amino_acids/2'&gt;active site amino acids&lt;/scene&gt;<br /> == Experimental Results/Function ==<br /> <br /> Once we felt confident enough to finalize our substrate hypothesis, we began testing in vitro. Beginning with bacterial protein overexpression and affinity chromatography, we were able to purify our POI and begin testing with real substrates. Below are the results of our Uncoupled Kinase Assay, reported in terms of specific activity (mg/mL). Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose. A total of five hexose substrates were tested in vitro, detailed in the table below. Based on these results, we were able to strengthen our initial hypothesis and continue characterization.<br /> <br /> [[Image:Specific_activity_table.png|400px|]]<br /> For further validation, we conducted an SDS analysis and provided below is the gel image. Indicated by the black box is our POI, around 34 kDa. Results were not as clear as anticipated, and in future studies, we would need to utilize different chromatography methods to yield higher quality protein concentrations and conduct a pre and post induction to visualize the purity of our protein.<br /> <br /> [[Image:FINAL_gel.png|250px|]]<br /> <br /> == Project Implications ==<br /> The goal of this project is to explore the techniques it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, including bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE. These techniques can be used to help characterize further putative kinases discovered in the future that do not have a defined function. This project is of importance because proteins are biomolecules responsible for as organisms' survival and understanding their unique function is essential for advances in modern medicine, scientific research, and agriculture.<br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> After various experiments and discussion, we have concluded that the novel protein 3R8E is a &lt;scene name='90/904995/Mesh_backbone_with_atp_glucose/3'&gt;Glucose Kinase&lt;/scene&gt;. By using an in-vitro assay, we obtained results comparing the phosphorylation rates of five sugars in the presence of our putative kinase. From the assay results, we were able to calculate an average specific activity for all experimental sugars, and by comparing these numbers we can clearly see that glucose is being phosphorylated in the presence of protein 3R8E. To get to this conclusion, we ran triplicates of the experiment for each sugar, and then followed a calculation procedure to get specific activity numbers to quantify how active our protein is with a given substrate. By doing this, we were able to validate and further support our hypothesis, which now can allow others to replicate or continue our research. Glucose provided us with a specific activity value of 0.214 +/- 0.893 and 1.223 +/- 2.575 for prep one and two respectively. <br /> <br /> Future Direction:<br /> <br /> After validating our results, we now look to take our findings to a micropublication website for undergraduate research. By doing this, not only will our work be forward facing and available to the science community, but it also allows for collaboration and further questions to be asked. After completing the micropublication, we look to continue to develop research strategies for putative kinases, as the PDB has thousands of proteins with unsolved functions. We will do this by combining machine learning, data science, and lab work to allow undergraduate students and scientist to effectively research and study putative kinase structures and functions. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3640504 2022-09-29T19:45:27Z

<p>Michel Evertsen: </p> <hr /> <div>==Characterization of the 3r8e Protein, a Novel Glucose Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, coupled kinase activity assays, and SDS PAGE analyses were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Previous and subsequent analysis of protein 3r8e validated our initial in silico and in vitro results. Overall, we have strong preliminary evidence that the our protein of interest (POI) is a glucose kinase.<br /> == Introduction ==<br /> As apart of a research project under the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) consortium, our group was tasked with characterizing and identifying the function of this protein to provide further insight of the protein's relationship to the bacteria. Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Previous research has shown that there is relationship between our POI and bacteria found in soil. Current research techniques have made the role more apparent and below is the general workflow detailing how we generated our conclusions.<br /> <br /> [[Image:Workflow1.png|500px|]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Structural and sequence alignment analysis with protein 3vov, a hexokinase, is provided below.<br /> <br /> [[Image:3R8EDali180.png|400px|]]<br /> [[Image:3R8EDALIHMM.png|400px|]] <br /> <br /> <br /> From here, we were able to form the conclusion that our POI interacts with glucose based on the alignment with a known hexokinase. To validate that glucose actually binds and interacts with our protein of interest, we conducted a PyRx in silico docking experiment with a total of five hexose substrates. Other substrates tested include fructose, galactose, lactose, and ribose, however, experimental in silico docking results for those substrates were significantly less than glucose. Along with the PyRx docking, we visualized ATP and glucose (Orange) within the proposed active site in the PyMol visualization below. The binding affinity of glucose was -5.1 kcal/mol, which strengthens our idea that glucose is phosphorylated by our protein of interest. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro and because ATP aids in the phosphorylation of glucose, an &lt;scene name='90/904995/3r8ec_w_glc_and_atp/1'&gt;interactive structure&lt;/scene&gt; has been provided to represent interactions of glucose and ATP in the active site.<br /> <br /> <br /> == Experimental Results/Function ==<br /> <br /> Once we felt confident enough to finalize our substrate hypothesis, we began testing in vitro. Beginning with bacterial protein overexpression and affinity chromatography, we were able to purify our POI and begin testing with real substrates. Below are the results of our Uncoupled Kinase Assay, reported in terms of specific activity (mg/mL). Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose. A total of five hexose substrates were tested in vitro, detailed in the table below. Based on these results, we were able to strengthen our initial hypothesis and continue characterization.<br /> <br /> [[Image:Specific_activity_table.png|400px|]]<br /> For further validation, we conducted an SDS analysis and provided below is the gel image. Indicated by the black box is our POI, around 34 kDa. Results were not as clear as anticipated, and in future studies, we would need to utilize different chromatography methods to yield higher quality protein concentrations and conduct a pre and post induction to visualize the purity of our protein.<br /> <br /> [[Image:FINAL_gel.png|250px|]]<br /> <br /> == Project Implications ==<br /> The goal of this project is to explore the techniques it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, including bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE. These techniques can be used to help characterize further putative kinases discovered in the future that do not have a defined function. This project is of importance because proteins are biomolecules responsible for as organisms' survival and understanding their unique function is essential for advances in modern medicine, scientific research, and agriculture.<br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> After various experiments and discussion, we have concluded that the novel protein 3R8E is a &lt;scene name='90/904995/Mesh_backbone_with_atp_glucose/3'&gt;Glucose Kinase&lt;/scene&gt;. By using an in-vitro assay, we obtained results comparing the phosphorylation rates of five sugars in the presence of our putative kinase. From the assay results, we were able to calculate an average specific activity for all experimental sugars, and by comparing these numbers we can clearly see that glucose is being phosphorylated in the presence of protein 3R8E. To get to this conclusion, we ran triplicates of the experiment for each sugar, and then followed a calculation procedure to get specific activity numbers to quantify how active our protein is with a given substrate. By doing this, we were able to validate and further support our hypothesis, which now can allow others to replicate or continue our research. Glucose provided us with a specific activity value of 0.214 +/- 0.893 and 1.223 +/- 2.575 for prep one and two respectively. <br /> <br /> Future Direction:<br /> <br /> After validating our results, we now look to take our findings to a micropublication website for undergraduate research. By doing this, not only will our work be forward facing and available to the science community, but it also allows for collaboration and further questions to be asked. After completing the micropublication, we look to continue to develop research strategies for putative kinases, as the PDB has thousands of proteins with unsolved functions. We will do this by combining machine learning, data science, and lab work to allow undergraduate students and scientist to effectively research and study putative kinase structures and functions. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3640503 2022-09-29T19:42:53Z

<p>Michel Evertsen: </p> <hr /> <div>==Characterization of the 3r8e Protein, a Novel Glucose Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, coupled kinase activity assays, and SDS PAGE analyses were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Previous and subsequent analysis of protein 3r8e validated our initial in silico and in vitro results. Overall, we have strong preliminary evidence that the our protein of interest (POI) is a glucose kinase.&lt;scene name='90/904995/Mesh_backbone_with_atp_glucose/3'&gt;Glucose Kinase&lt;/scene&gt;<br /> == Introduction ==<br /> As apart of a research project under the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) consortium, our group was tasked with characterizing and identifying the function of this protein to provide further insight of the protein's relationship to the bacteria. Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Previous research has shown that there is relationship between our POI and bacteria found in soil. Current research techniques have made the role more apparent and below is the general workflow detailing how we generated our conclusions.<br /> <br /> [[Image:Workflow1.png|500px|]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Structural and sequence alignment analysis with protein 3vov, a hexokinase, is provided below.<br /> <br /> [[Image:3R8EDali180.png|400px|]]<br /> [[Image:3R8EDALIHMM.png|400px|]] <br /> <br /> <br /> From here, we were able to form the conclusion that our POI interacts with glucose based on the alignment with a known hexokinase. To validate that glucose actually binds and interacts with our protein of interest, we conducted a PyRx in silico docking experiment with a total of five hexose substrates. Other substrates tested include fructose, galactose, lactose, and ribose, however, experimental in silico docking results for those substrates were significantly less than glucose. Along with the PyRx docking, we visualized ATP and glucose (Orange) within the proposed active site in the PyMol visualization below. The binding affinity of glucose was -5.1 kcal/mol, which strengthens our idea that glucose is phosphorylated by our protein of interest. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro and because ATP aids in the phosphorylation of glucose, an &lt;scene name='90/904995/3r8ec_w_glc_and_atp/1'&gt;interactive structure&lt;/scene&gt; has been provided to represent interactions of glucose and ATP in the active site.<br /> <br /> <br /> == Experimental Results/Function ==<br /> <br /> Once we felt confident enough to finalize our substrate hypothesis, we began testing in vitro. Beginning with bacterial protein overexpression and affinity chromatography, we were able to purify our POI and begin testing with real substrates. Below are the results of our Uncoupled Kinase Assay, reported in terms of specific activity (mg/mL). Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose. A total of five hexose substrates were tested in vitro, detailed in the table below. Based on these results, we were able to strengthen our initial hypothesis and continue characterization.<br /> <br /> [[Image:Specific_activity_table.png|400px|]]<br /> For further validation, we conducted an SDS analysis and provided below is the gel image. Indicated by the black box is our POI, around 34 kDa. Results were not as clear as anticipated, and in future studies, we would need to utilize different chromatography methods to yield higher quality protein concentrations and conduct a pre and post induction to visualize the purity of our protein.<br /> <br /> [[Image:FINAL_gel.png|250px|]]<br /> <br /> == Project Implications ==<br /> The goal of this project is to explore the techniques it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, including bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE. These techniques can be used to help characterize further putative kinases discovered in the future that do not have a defined function. This project is of importance because proteins are biomolecules responsible for as organisms' survival and understanding their unique function is essential for advances in modern medicine, scientific research, and agriculture.<br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> After various experiments and discussion, we have concluded that the novel protein 3R8E is a &lt;scene name='90/904995/Mesh_backbone_with_atp_glucose/3'&gt;Glucose Kinase&lt;/scene&gt;. By using an in-vitro assay, we obtained results comparing the phosphorylation rates of five sugars in the presence of our putative kinase. From the assay results, we were able to calculate an average specific activity for all experimental sugars, and by comparing these numbers we can clearly see that glucose is being phosphorylated in the presence of protein 3R8E. To get to this conclusion, we ran triplicates of the experiment for each sugar, and then followed a calculation procedure to get specific activity numbers to quantify how active our protein is with a given substrate. By doing this, we were able to validate and further support our hypothesis, which now can allow others to replicate or continue our research. Glucose provided us with a specific activity value of 0.214 +/- 0.893 and 1.223 +/- 2.575 for prep one and two respectively. <br /> <br /> Future Direction:<br /> <br /> After validating our results, we now look to take our findings to a micropublication website for undergraduate research. By doing this, not only will our work be forward facing and available to the science community, but it also allows for collaboration and further questions to be asked. After completing the micropublication, we look to continue to develop research strategies for putative kinases, as the PDB has thousands of proteins with unsolved functions. We will do this by combining machine learning, data science, and lab work to allow undergraduate students and scientist to effectively research and study putative kinase structures and functions. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3640502 2022-09-29T19:38:32Z

<p>Michel Evertsen: </p> <hr /> <div>==Characterization of the 3r8e Protein, a Novel Glucose Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, coupled kinase activity assays, and SDS PAGE analyses were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Previous and subsequent analysis of protein 3r8e validated our initial in silico and in vitro results. Overall, we have strong preliminary evidence that the our protein of interest (POI) is a &lt;scene name='90/904995/Mesh_backbone_with_atp_glucose/3'&gt;Glucose Kinase&lt;/scene&gt;.<br /> == Introduction ==<br /> As apart of a research project under the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) consortium, our group was tasked with characterizing and identifying the function of this protein to provide further insight of the protein's relationship to the bacteria. Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Previous research has shown that there is relationship between our POI and bacteria found in soil. Current research techniques have made the role more apparent and below is the general workflow detailing how we generated our conclusions.<br /> <br /> [[Image:Workflow1.png|500px|]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Structural and sequence alignment analysis with protein 3vov, a hexokinase, is provided below.<br /> <br /> [[Image:3R8EDali180.png|400px|]]<br /> [[Image:3R8EDALIHMM.png|400px|]] <br /> <br /> <br /> From here, we were able to form the conclusion that our POI interacts with glucose based on the alignment with a known hexokinase. To validate that glucose actually binds and interacts with our protein of interest, we conducted a PyRx in silico docking experiment with a total of five hexose substrates. Other substrates tested include fructose, galactose, lactose, and ribose, however, experimental in silico docking results for those substrates were significantly less than glucose. Along with the PyRx docking, we visualized ATP and glucose (Orange) within the proposed active site in the PyMol visualization below. The binding affinity of glucose was -5.1 kcal/mol, which strengthens our idea that glucose is phosphorylated by our protein of interest. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro and because ATP aids in the phosphorylation of glucose, an &lt;scene name='90/904995/3r8ec_w_glc_and_atp/1'&gt;interactive structure&lt;/scene&gt; has been provided to represent interactions of glucose and ATP in the active site.<br /> <br /> <br /> == Experimental Results/Function ==<br /> <br /> Once we felt confident enough to finalize our substrate hypothesis, we began testing in vitro. Beginning with bacterial protein overexpression and affinity chromatography, we were able to purify our POI and begin testing with real substrates. Below are the results of our Uncoupled Kinase Assay, reported in terms of specific activity (mg/mL). Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose. A total of five hexose substrates were tested in vitro, detailed in the table below. Based on these results, we were able to strengthen our initial hypothesis and continue characterization.<br /> <br /> [[Image:Specific_activity_table.png|400px|]]<br /> For further validation, we conducted an SDS analysis and provided below is the gel image. Indicated by the black box is our POI, around 34 kDa. Results were not as clear as anticipated, and in future studies, we would need to utilize different chromatography methods to yield higher quality protein concentrations and conduct a pre and post induction to visualize the purity of our protein.<br /> <br /> [[Image:FINAL_gel.png|250px|]]<br /> <br /> == Project Implications ==<br /> The goal of this project is to explore the techniques it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, including bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE. These techniques can be used to help characterize further putative kinases discovered in the future that do not have a defined function. This project is of importance because proteins are biomolecules responsible for as organisms' survival and understanding their unique function is essential for advances in modern medicine, scientific research, and agriculture.<br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> After various experiments and discussion, we have concluded that the novel protein 3R8E is a glucose kinase. By using an in-vitro assay, we obtained results comparing the phosphorylation rates of five sugars in the presence of our putative kinase. From the assay results, we were able to calculate an average specific activity for all experimental sugars, and by comparing these numbers we can clearly see that glucose is being phosphorylated in the presence of protein 3R8E. To get to this conclusion, we ran triplicates of the experiment for each sugar, and then followed a calculation procedure to get specific activity numbers to quantify how active our protein is with a given substrate. By doing this, we were able to validate and further support our hypothesis, which now can allow others to replicate or continue our research. Glucose provided us with a specific activity value of 0.214 +/- 0.893 and 1.223 +/- 2.575 for prep one and two respectively. <br /> <br /> Future Direction:<br /> <br /> After validating our results, we now look to take our findings to a micropublication website for undergraduate research. By doing this, not only will our work be forward facing and available to the science community, but it also allows for collaboration and further questions to be asked. After completing the micropublication, we look to continue to develop research strategies for putative kinases, as the PDB has thousands of proteins with unsolved functions. We will do this by combining machine learning, data science, and lab work to allow undergraduate students and scientist to effectively research and study putative kinase structures and functions. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3640501 2022-09-29T19:37:42Z

<p>Michel Evertsen: </p> <hr /> <div>==Characterization of the 3r8e Protein, a Novel Glucose Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, coupled kinase activity assays, and SDS PAGE analyses were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Previous and subsequent analysis of protein 3r8e validated our initial in silico and in vitro results. Overall, we have strong preliminary evidence that the our protein of interest (POI) is a&lt;scene name='90/904995/Mesh_backbone_with_atp_glucose/3'&gt;Glucose Kinase&lt;/scene&gt;.<br /> == Introduction ==<br /> As apart of a research project under the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) consortium, our group was tasked with characterizing and identifying the function of this protein to provide further insight of the protein's relationship to the bacteria. Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Previous research has shown that there is relationship between our POI and bacteria found in soil. Current research techniques have made the role more apparent and below is the general workflow detailing how we generated our conclusions.<br /> <br /> [[Image:Workflow1.png|500px|]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Structural and sequence alignment analysis with protein 3vov, a hexokinase, is provided below.<br /> <br /> [[Image:3R8EDali180.png|400px|]]<br /> [[Image:3R8EDALIHMM.png|400px|]] <br /> <br /> <br /> From here, we were able to form the conclusion that our POI interacts with glucose based on the alignment with a known hexokinase. To validate that glucose actually binds and interacts with our protein of interest, we conducted a PyRx in silico docking experiment with a total of five hexose substrates. Other substrates tested include fructose, galactose, lactose, and ribose, however, experimental in silico docking results for those substrates were significantly less than glucose. Along with the PyRx docking, we visualized ATP and glucose (Orange) within the proposed active site in the PyMol visualization below. The binding affinity of glucose was -5.1 kcal/mol, which strengthens our idea that glucose is phosphorylated by our protein of interest. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro and because ATP aids in the phosphorylation of glucose, an &lt;scene name='90/904995/3r8ec_w_glc_and_atp/1'&gt;interactive structure&lt;/scene&gt; has been provided to represent interactions of glucose and ATP in the active site.<br /> <br /> <br /> == Experimental Results/Function ==<br /> <br /> Once we felt confident enough to finalize our substrate hypothesis, we began testing in vitro. Beginning with bacterial protein overexpression and affinity chromatography, we were able to purify our POI and begin testing with real substrates. Below are the results of our Uncoupled Kinase Assay, reported in terms of specific activity (mg/mL). Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose. A total of five hexose substrates were tested in vitro, detailed in the table below. Based on these results, we were able to strengthen our initial hypothesis and continue characterization.<br /> <br /> [[Image:Specific_activity_table.png|400px|]]<br /> For further validation, we conducted an SDS analysis and provided below is the gel image. Indicated by the black box is our POI, around 34 kDa. Results were not as clear as anticipated, and in future studies, we would need to utilize different chromatography methods to yield higher quality protein concentrations and conduct a pre and post induction to visualize the purity of our protein.<br /> <br /> [[Image:FINAL_gel.png|250px|]]<br /> <br /> == Project Implications ==<br /> The goal of this project is to explore the techniques it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, including bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE. These techniques can be used to help characterize further putative kinases discovered in the future that do not have a defined function. This project is of importance because proteins are biomolecules responsible for as organisms' survival and understanding their unique function is essential for advances in modern medicine, scientific research, and agriculture.<br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> After various experiments and discussion, we have concluded that the novel protein 3R8E is a glucose kinase. By using an in-vitro assay, we obtained results comparing the phosphorylation rates of five sugars in the presence of our putative kinase. From the assay results, we were able to calculate an average specific activity for all experimental sugars, and by comparing these numbers we can clearly see that glucose is being phosphorylated in the presence of protein 3R8E. To get to this conclusion, we ran triplicates of the experiment for each sugar, and then followed a calculation procedure to get specific activity numbers to quantify how active our protein is with a given substrate. By doing this, we were able to validate and further support our hypothesis, which now can allow others to replicate or continue our research. Glucose provided us with a specific activity value of 0.214 +/- 0.893 and 1.223 +/- 2.575 for prep one and two respectively. <br /> <br /> Future Direction:<br /> <br /> After validating our results, we now look to take our findings to a micropublication website for undergraduate research. By doing this, not only will our work be forward facing and available to the science community, but it also allows for collaboration and further questions to be asked. After completing the micropublication, we look to continue to develop research strategies for putative kinases, as the PDB has thousands of proteins with unsolved functions. We will do this by combining machine learning, data science, and lab work to allow undergraduate students and scientist to effectively research and study putative kinase structures and functions. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3640500 2022-09-29T19:28:18Z

<p>Michel Evertsen: </p> <hr /> <div>==Characterization of the 3r8e Protein, a Novel Glucose Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, coupled kinase activity assays, and SDS PAGE analyses were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Previous and subsequent analysis of protein 3r8e validated our initial in silico and in vitro results. Overall, we have strong preliminary evidence that the our protein of interest (POI) is a &lt;scene name='90/904995/Mesh_backbone_with_atp_glucose/2'&gt;glucose kinase&lt;/scene&gt;.<br /> == Introduction ==<br /> As apart of a research project under the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) consortium, our group was tasked with characterizing and identifying the function of this protein to provide further insight of the protein's relationship to the bacteria. Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Previous research has shown that there is relationship between our POI and bacteria found in soil. Current research techniques have made the role more apparent and below is the general workflow detailing how we generated our conclusions.<br /> <br /> [[Image:Workflow1.png|500px|]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Structural and sequence alignment analysis with protein 3vov, a hexokinase, is provided below.<br /> <br /> [[Image:3R8EDali180.png|400px|]]<br /> [[Image:3R8EDALIHMM.png|400px|]] <br /> <br /> <br /> From here, we were able to form the conclusion that our POI interacts with glucose based on the alignment with a known hexokinase. To validate that glucose actually binds and interacts with our protein of interest, we conducted a PyRx in silico docking experiment with a total of five hexose substrates. Other substrates tested include fructose, galactose, lactose, and ribose, however, experimental in silico docking results for those substrates were significantly less than glucose. Along with the PyRx docking, we visualized ATP and glucose (Orange) within the proposed active site in the PyMol visualization below. The binding affinity of glucose was -5.1 kcal/mol, which strengthens our idea that glucose is phosphorylated by our protein of interest. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro and because ATP aids in the phosphorylation of glucose, an &lt;scene name='90/904995/3r8ec_w_glc_and_atp/1'&gt;interactive structure&lt;/scene&gt; has been provided to represent interactions of glucose and ATP in the active site.<br /> <br /> <br /> == Experimental Results/Function ==<br /> <br /> Once we felt confident enough to finalize our substrate hypothesis, we began testing in vitro. Beginning with bacterial protein overexpression and affinity chromatography, we were able to purify our POI and begin testing with real substrates. Below are the results of our Uncoupled Kinase Assay, reported in terms of specific activity (mg/mL). Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose. A total of five hexose substrates were tested in vitro, detailed in the table below. Based on these results, we were able to strengthen our initial hypothesis and continue characterization.<br /> <br /> [[Image:Specific_activity_table.png|400px|]]<br /> For further validation, we conducted an SDS analysis and provided below is the gel image. Indicated by the black box is our POI, around 34 kDa. Results were not as clear as anticipated, and in future studies, we would need to utilize different chromatography methods to yield higher quality protein concentrations and conduct a pre and post induction to visualize the purity of our protein.<br /> <br /> [[Image:FINAL_gel.png|250px|]]<br /> <br /> == Project Implications ==<br /> The goal of this project is to explore the techniques it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, including bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE. These techniques can be used to help characterize further putative kinases discovered in the future that do not have a defined function. This project is of importance because proteins are biomolecules responsible for as organisms' survival and understanding their unique function is essential for advances in modern medicine, scientific research, and agriculture.<br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> After various experiments and discussion, we have concluded that the novel protein 3R8E is a glucose kinase. By using an in-vitro assay, we obtained results comparing the phosphorylation rates of five sugars in the presence of our putative kinase. From the assay results, we were able to calculate an average specific activity for all experimental sugars, and by comparing these numbers we can clearly see that glucose is being phosphorylated in the presence of protein 3R8E. To get to this conclusion, we ran triplicates of the experiment for each sugar, and then followed a calculation procedure to get specific activity numbers to quantify how active our protein is with a given substrate. By doing this, we were able to validate and further support our hypothesis, which now can allow others to replicate or continue our research. Glucose provided us with a specific activity value of 0.214 +/- 0.893 and 1.223 +/- 2.575 for prep one and two respectively. <br /> <br /> Future Direction:<br /> <br /> After validating our results, we now look to take our findings to a micropublication website for undergraduate research. By doing this, not only will our work be forward facing and available to the science community, but it also allows for collaboration and further questions to be asked. After completing the micropublication, we look to continue to develop research strategies for putative kinases, as the PDB has thousands of proteins with unsolved functions. We will do this by combining machine learning, data science, and lab work to allow undergraduate students and scientist to effectively research and study putative kinase structures and functions. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3640499 2022-09-29T19:25:26Z

<p>Michel Evertsen: </p> <hr /> <div>==Characterization of the 3r8e Protein, a Novel Glucose Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, coupled kinase activity assays, and SDS PAGE analyses were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Previous and subsequent analysis of protein 3r8e validated our initial in silico and in vitro results. Overall, we have strong preliminary evidence that the our protein of interest (POI) is a glucose kinase.<br /> == Introduction ==<br /> As apart of a research project under the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) consortium, our group was tasked with characterizing and identifying the function of this protein to provide further insight of the protein's relationship to the bacteria. Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Previous research has shown that there is relationship between our POI and bacteria found in soil. Current research techniques have made the role more apparent and below is the general workflow detailing how we generated our conclusions.<br /> <br /> [[Image:Workflow1.png|500px|]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Structural and sequence alignment analysis with protein 3vov, a hexokinase, is provided below.<br /> <br /> [[Image:3R8EDali180.png|400px|]]<br /> [[Image:3R8EDALIHMM.png|400px|]] <br /> <br /> &lt;scene name='90/904995/Mesh_backbone_with_atp_glucose/1'&gt;Text To Be Displayed&lt;/scene&gt;<br /> From here, we were able to form the conclusion that our POI interacts with glucose based on the alignment with a known hexokinase. To validate that glucose actually binds and interacts with our protein of interest, we conducted a PyRx in silico docking experiment with a total of five hexose substrates. Other substrates tested include fructose, galactose, lactose, and ribose, however, experimental in silico docking results for those substrates were significantly less than glucose. Along with the PyRx docking, we visualized ATP and glucose (Orange) within the proposed active site in the PyMol visualization below. The binding affinity of glucose was -5.1 kcal/mol, which strengthens our idea that glucose is phosphorylated by our protein of interest. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro and because ATP aids in the phosphorylation of glucose, an &lt;scene name='90/904995/3r8ec_w_glc_and_atp/1'&gt;interactive structure&lt;/scene&gt; has been provided to represent interactions of glucose and ATP in the active site.<br /> <br /> <br /> == Experimental Results/Function ==<br /> <br /> Once we felt confident enough to finalize our substrate hypothesis, we began testing in vitro. Beginning with bacterial protein overexpression and affinity chromatography, we were able to purify our POI and begin testing with real substrates. Below are the results of our Uncoupled Kinase Assay, reported in terms of specific activity (mg/mL). Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose. A total of five hexose substrates were tested in vitro, detailed in the table below. Based on these results, we were able to strengthen our initial hypothesis and continue characterization.<br /> <br /> [[Image:Specific_activity_table.png|400px|]]<br /> For further validation, we conducted an SDS analysis and provided below is the gel image. Indicated by the black box is our POI, around 34 kDa. Results were not as clear as anticipated, and in future studies, we would need to utilize different chromatography methods to yield higher quality protein concentrations and conduct a pre and post induction to visualize the purity of our protein.<br /> <br /> [[Image:FINAL_gel.png|250px|]]<br /> <br /> == Project Implications ==<br /> The goal of this project is to explore the techniques it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, including bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE. These techniques can be used to help characterize further putative kinases discovered in the future that do not have a defined function. This project is of importance because proteins are biomolecules responsible for as organisms' survival and understanding their unique function is essential for advances in modern medicine, scientific research, and agriculture.<br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> After various experiments and discussion, we have concluded that the novel protein 3R8E is a glucose kinase. By using an in-vitro assay, we obtained results comparing the phosphorylation rates of five sugars in the presence of our putative kinase. From the assay results, we were able to calculate an average specific activity for all experimental sugars, and by comparing these numbers we can clearly see that glucose is being phosphorylated in the presence of protein 3R8E. To get to this conclusion, we ran triplicates of the experiment for each sugar, and then followed a calculation procedure to get specific activity numbers to quantify how active our protein is with a given substrate. By doing this, we were able to validate and further support our hypothesis, which now can allow others to replicate or continue our research. Glucose provided us with a specific activity value of 0.214 +/- 0.893 and 1.223 +/- 2.575 for prep one and two respectively. <br /> <br /> Future Direction:<br /> <br /> After validating our results, we now look to take our findings to a micropublication website for undergraduate research. By doing this, not only will our work be forward facing and available to the science community, but it also allows for collaboration and further questions to be asked. After completing the micropublication, we look to continue to develop research strategies for putative kinases, as the PDB has thousands of proteins with unsolved functions. We will do this by combining machine learning, data science, and lab work to allow undergraduate students and scientist to effectively research and study putative kinase structures and functions. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=File:GLCconf_WithATP-and-2-more.pdb&diff=3640498 2022-09-29T18:56:45Z

<p>Michel Evertsen: For PDB page of 3R8E</p> <hr /> <div>For PDB page of 3R8E</div>

Michel Evertsen https://proteopedia.org/index.php?title=File:GLCconf_WithATP.pdb&diff=3640496 2022-09-29T18:53:10Z

<p>Michel Evertsen: </p> <hr /> <div></div>

Michel Evertsen https://proteopedia.org/index.php?title=File:3r8e-Clean.pdb&diff=3640495 2022-09-29T18:49:25Z

<p>Michel Evertsen: </p> <hr /> <div></div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3640471 2022-09-28T19:38:40Z

<p>Michel Evertsen: </p> <hr /> <div>==Characterization of the 3r8e Protein, a Novel Glucose Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, coupled kinase activity assays, and SDS PAGE analyses were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Previous and subsequent analysis of protein 3r8e validated our initial in silico and in vitro results. Overall, we have strong preliminary evidence that the our protein of interest (POI) is a glucose kinase.<br /> == Introduction ==<br /> As apart of a research project under the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) consortium, our group was tasked with characterizing and identifying the function of this protein to provide further insight of the protein's relationship to the bacteria. Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Previous research has shown that there is relationship between our POI and bacteria found in soil. Current research techniques have made the role more apparent and below is the general workflow detailing how we generated our conclusions.<br /> <br /> [[Image:Workflow1.png|500px|]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Structural and sequence alignment analysis with protein 3vov, a hexokinase, is provided below.<br /> <br /> [[Image:3R8EDali180.png|400px|]]<br /> [[Image:3R8EDALIHMM.png|400px|]] <br /> <br /> With this binding affinity in mind, we visualized the active site interactions amongst our POI, glucose, and ATP. To do this, we used PyMol to generate a mesh view of our protein, below, and docked glucose (yellow) and ATP (blue) in the proposed active site. These results, along with supporting evidence from the previous in silico tools, allowed us to continue testing our hypothesis with confidence.<br /> <br /> [[Image:MeshviewProteinW_atp_glc.png|px|200]]<br /> <br /> To validate that glucose actually binds and interacts with our protein of interest, we conducted a PyRx in silico docking experiment with a total of five hexose substrates. Other substrates tested include fructose, galactose, lactose, and ribose, however, experimental in silico docking results for those substrates were significantly less than glucose. The binding affinity of glucose was -5.1 kcal/mol, which strengthens our idea that glucose is phosphorylated by our protein of interest. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro and because ATP is a commonality amongst sugar kinases, an &lt;scene name='90/904995/3r8ec_w_glc_and_atp/1'&gt;interactive structure&lt;/scene&gt; has been provided to represent interactions of glucose and ATP in the active site.<br /> <br /> == Experimental Results/Function ==<br /> <br /> Once we felt confident enough to finalize our substrate hypothesis, we began testing in vitro. Beginning with bacterial protein overexpression and affinity chromatography, we were able to purify our POI and begin testing with real substrates. Below are the results of our Uncoupled Kinase Assay, reported in terms of specific activity (mg/mL). Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose. A total of five hexose substrates were tested in vitro, detailed in the table below. Based on these results, we were able to strengthen our initial hypothesis and continue characterization.<br /> <br /> [[Image:Specific_activity_table.png|400px|]]<br /> For further validation, we conducted an SDS analysis and provided below is the gel image. Indicated by the black box is our POI, around 34 kDa. Results were not as clear as anticipated, and in future studies, we would need to utilize different chromatography methods to yield higher quality protein concentrations and conduct a pre and post induction to visualize the purity of our protein.<br /> <br /> [[Image:FINAL_gel.png|250px|]]<br /> <br /> == Project Implications ==<br /> The goal of this project is to explore the techniques it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, including bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE. These techniques can be used to help characterize further putative kinases discovered in the future that do not have a defined function. This project is of importance because proteins are biomolecules responsible for as organisms' survival and understanding their unique function is essential for advances in modern medicine, scientific research, and agriculture.<br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> After various experiments and discussion, we have concluded that the novel protein 3R8E is a glucose kinase. By using an in-vitro assay, we obtained results comparing the phosphorylation rates of five sugars in the presence of our putative kinase. From the assay results, we were able to calculate an average specific activity for all experimental sugars, and by comparing these numbers we can clearly see that glucose is being phosphorylated in the presence of protein 3R8E. To get to this conclusion, we ran triplicates of the experiment for each sugar, and then followed a calculation procedure to get specific activity numbers to quantify how active our protein is with a given substrate. By doing this, we were able to validate and further support our hypothesis, which now can allow others to replicate or continue our research. Glucose provided us with a specific activity value of 0.214 +/- 0.893 and 1.223 +/- 2.575 for prep one and two respectively. <br /> <br /> Future Direction:<br /> <br /> After validating our results, we now look to take our findings to a micropublication website for undergraduate research. By doing this, not only will our work be forward facing and available to the science community, but it also allows for collaboration and further questions to be asked. After completing the micropublication, we look to continue to develop research strategies for putative kinases, as the PDB has thousands of proteins with unsolved functions. We will do this by combining machine learning, data science, and lab work to allow undergraduate students and scientist to effectively research and study putative kinase structures and functions. <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3640470 2022-09-28T19:34:10Z

<p>Michel Evertsen: </p> <hr /> <div>==Characterization of the 3r8e Protein, a Novel Glucose Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, coupled kinase activity assays, and SDS PAGE analyses were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Previous and subsequent analysis of protein 3r8e validated our initial in silico and in vitro results. Overall, we have strong preliminary evidence that the our protein of interest (POI) is a glucose kinase.<br /> == Introduction ==<br /> As apart of a research project under the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) consortium, our group was tasked with characterizing and identifying the function of this protein to provide further insight of the protein's relationship to the bacteria. Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Previous research has shown that there is relationship between our POI and bacteria found in soil. Current research techniques have made the role more apparent and below is the general workflow detailing how we generated our conclusions.<br /> <br /> [[Image:Workflow1.png|500px|]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Structural and sequence alignment analysis with protein 3vov, a hexokinase, is provided below.<br /> <br /> [[Image:3R8EDali180.png|400px|]]<br /> [[Image:3R8EDALIHMM.png|400px|]] <br /> <br /> With this binding affinity in mind, we visualized the active site interactions amongst our POI, glucose, and ATP. To do this, we used PyMol to generate a mesh view of our protein, below, and docked glucose (yellow) and ATP (blue) in the proposed active site. These results, along with supporting evidence from the previous in silico tools, allowed us to continue testing our hypothesis with confidence.<br /> <br /> [[Image:MeshviewProteinW_atp_glc.png|px|200]]<br /> <br /> To validate that glucose actually binds and interacts with our protein of interest, we conducted a PyRx in silico docking experiment with a total of five hexose substrates. Other substrates tested include fructose, galactose, lactose, and ribose, however, experimental in silico docking results for those substrates were significantly less than glucose. The binding affinity of glucose was -5.1 kcal/mol, which strengthens our idea that glucose is phosphorylated by our protein of interest. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro and because ATP is a commonality amongst sugar kinases, an &lt;scene name='90/904995/3r8ec_w_glc_and_atp/1'&gt;interactive structure&lt;/scene&gt; has been provided to represent interactions of glucose and ATP in the active site.<br /> <br /> == Experimental Results/Function ==<br /> <br /> Once we felt confident enough to finalize our substrate hypothesis, we began testing in vitro. Beginning with bacterial protein overexpression and affinity chromatography, we were able to purify our POI and begin testing with real substrates. Below are the results of our Uncoupled Kinase Assay, reported in terms of specific activity (mg/mL). Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose. A total of five hexose substrates were tested in vitro, detailed in the table below. Based on these results, we were able to strengthen our initial hypothesis and continue characterization.<br /> <br /> [[Image:Specific_activity_table.png|400px|]]<br /> For further validation, we conducted an SDS analysis and provided below is the gel image. Indicated by the black box is our POI, around 34 kDa. Results were not as clear as anticipated, and in future studies, we would need to utilize different chromatography methods to yield higher quality protein concentrations and conduct a pre and post induction to visualize the purity of our protein.<br /> <br /> [[Image:FINAL_gel.png|250px|]]<br /> <br /> == Project Implications ==<br /> The goal of this project is to explore the techniques it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, including bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE. These techniques can be used to help characterize further putative kinases discovered in the future that do not have a defined function. This project is of importance because proteins are biomolecules responsible for as organisms' survival and understanding their unique function is essential for advances in modern medicine, scientific research, and agriculture.<br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> After various experiments and discussion, we have concluded that the novel protein 3R8E is a glucose kinase. By using an in-vitro assay, we obtained results comparing the phosphorylation rates of five sugars in the presence of our putative kinase. From the assay results. we were able to calculate an average specific activity for all experimental sugars, and by comparing these numbers we can clearly see that glucose is being phosphorylated in the presence of protein 3R8E. To get to this conclusion, we ran triplicates of the experiment for each sugar, and then followed a calculation procedure to get specific activity numbers to quantify how active our protein is with a given substrate. By doing this, we were able to validate and further support our hypothesis, which now can allow others to replicate or continue our research. <br /> <br /> Future Direction:<br /> <br /> After validating our results, we now look to take our findings to a micropublication website for undergraduate research. By doing this, not only will our work be forward facing and available to the science community, but it also allows for collaboration and further questions to be asked. After completing the micropublication, we look to continue to develop research strategies for putative kinases, as the PDB has thousands of proteins with unsolved functions. We will do this by combining machine learning, data science, and lab work to allow undergraduate students and scientist to effectively research and study putative kinase structures and functions. <br /> <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3640469 2022-09-28T19:15:44Z

<p>Michel Evertsen: </p> <hr /> <div>==Characterization of the 3r8e Protein, a Novel Glucose Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, coupled kinase activity assays, and SDS PAGE analyses were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Previous and subsequent analysis of protein 3r8e validated our initial in silico and in vitro results. Overall, we have strong preliminary evidence that the our protein of interest (POI) is a glucose kinase.<br /> == Introduction ==<br /> As apart of a research project under the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) consortium, our group was tasked with characterizing and identifying the function of this protein to provide further insight of the protein's relationship to the bacteria. Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Previous research has shown that there is relationship between our POI and bacteria found in soil. Current research techniques have made the role more apparent and below is the general workflow detailing how we generated our conclusions.<br /> <br /> [[Image:Workflow1.png|500px|]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Structural and sequence alignment analysis with protein 3vov, a hexokinase, is provided below.<br /> <br /> [[Image:3R8EDali180.png|400px|]]<br /> [[Image:3R8EDALIHMM.png|400px|]] <br /> <br /> With this binding affinity in mind, we visualized the active site interactions amongst our POI, glucose, and ATP. To do this, we used PyMol to generate a mesh view of our protein, below, and docked glucose (yellow) and ATP (blue) in the proposed active site. These results, along with supporting evidence from the previous in silico tools, allowed us to continue testing our hypothesis with confidence.<br /> <br /> [[Image:MeshviewProteinW_atp_glc.png|px|200]]<br /> <br /> To validate that glucose actually binds and interacts with our protein of interest, we conducted a PyRx in silico docking experiment with a total of five hexose substrates. Other substrates tested include fructose, galactose, lactose, and ribose, however, experimental in silico docking results for those substrates were significantly less than glucose. The binding affinity of glucose was -5.1 kcal/mol, which strengthens our idea that glucose is phosphorylated by our protein of interest. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro and because ATP is a commonality amongst sugar kinases, an &lt;scene name='90/904995/3r8ec_w_glc_and_atp/1'&gt;interactive structure&lt;/scene&gt; has been provided to represent interactions of glucose and ATP in the active site.<br /> <br /> == Experimental Results/Function ==<br /> <br /> Once we felt confident enough to finalize our substrate hypothesis, we began testing in vitro. Beginning with bacterial protein overexpression and affinity chromatography, we were able to purify our POI and begin testing with real substrates. Below are the results of our Uncoupled Kinase Assay, reported in terms of specific activity (mg/mL). Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose. A total of five hexose substrates were tested in vitro, detailed in the table below. Based on these results, we were able to strengthen our initial hypothesis and continue characterization.<br /> <br /> [[Image:Specific_activity_table.png|400px|]]<br /> For further validation, we conducted an SDS analysis and provided below is the gel image. Indicated by the black box is our POI, around 34 kDa. Results were not as clear as anticipated, and in future studies, we would need to utilize different chromatography methods to yield higher quality protein concentrations and conduct a pre and post induction to visualize the purity of our protein.<br /> <br /> [[Image:FINAL_gel.png|250px|]]<br /> <br /> == Project Implications ==<br /> The goal of this project is to explore the techniques it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, including bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE. These techniques can be used to help characterize further putative kinases discovered in the future that do not have a defined function. This project is of importance because proteins are biomolecules responsible for as organisms' survival and understanding their unique function is essential for advances in modern medicine, scientific research, and agriculture.<br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> After various experiments and discussion, we have concluded that the novel protein 3R8E is a glucose kinase. By using an in-vitro assay, we obtained results comparing the phosphorylation rates of five sugars in the presence of our putative kinase. From the assay results. we were able to calculate an average specific activity for all experimental sugars, and by comparing these numbers we can clearly see that glucose is being phosphorylated in the presence of protein 3R8E (insert table of results?). <br /> <br /> To get to this conclusion, we completed and compared different assay trial results. In each assay, we ran triplicates of the experiment for each sugar, and then followed a calculation procedure to get specific activity numbers to quantify how active our protein is with a given substrate. <br /> <br /> <br /> - D-Glucose exhibited a specific activity of ___ ug/mL<br /> <br /> - Specific activity: D-Glucose &gt;&gt; D-Galactose &gt; D-Fructose &gt; Lactose &gt; D-Ribose<br /> <br /> <br /> <br /> Future Direction:<br /> <br /> - Publish results for further application<br /> <br /> <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3636195 2022-09-28T01:35:20Z

<p>Michel Evertsen: </p> <hr /> <div>==Characterization of the 3r8e Protein, a Novel Glucose Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, coupled kinase activity assays, and SDS PAGE analyses were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Previous and subsequent analysis of protein 3r8e validated our initial in silico and in vitro results. Overall, we have strong preliminary evidence that the our protein of interest (POI) is a glucose kinase.<br /> == Introduction ==<br /> As apart of a research project under the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) consortium, our group was tasked with characterizing and identifying the function of this protein to provide further insight of the protein's relationship to the bacteria. Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Previous research has shown that there is relationship between our POI and bacteria found in soil. Current research techniques have made the role more apparent and below is the general workflow detailing how we generated our conclusions.<br /> <br /> [[Image:Workflow1.png|500px|]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Structural and sequence alignment analysis with protein 3vov, a hexokinase, is provided below.<br /> <br /> [[Image:3R8EDali180.png|400px|]]<br /> [[Image:3R8EDALIHMM.png|400px|]] <br /> <br /> With this binding affinity in mind, we visualized the active site interactions amongst our POI, glucose, and ATP. To do this, we used PyMol to generate a mesh view of our protein, below, and docked glucose (yellow) and ATP (blue) in the proposed active site. These results, along with supporting evidence from the previous in silico tools, allowed us to continue testing our hypothesis with confidence.<br /> <br /> [[Image:MeshviewProteinW_atp_glc.png|px|200]]<br /> <br /> To validate that glucose actually binds and interacts with our protein of interest, we conducted a PyRx in silico docking experiment with a total of five hexose substrates. Other substrates tested include fructose, galactose, lactose, and ribose, however, experimental in silico docking results for those substrates were significantly less than glucose. The binding affinity of glucose was -5.1 kcal/mol, which strengthens our idea that glucose is phosphorylated by our protein of interest. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro and because ATP is a commonality amongst sugar kinases, an &lt;scene name='90/904995/3r8ec_w_glc_and_atp/1'&gt;interactive structure&lt;/scene&gt; has been provided to represent interactions of glucose and ATP in the active site.<br /> <br /> == Experimental Results/Function ==<br /> <br /> Once we felt confident enough to finalize our substrate hypothesis, we began testing in vitro. Beginning with bacterial protein overexpression and affinity chromatography, we were able to purify our POI and begin testing with real substrates. Below are the results of our Uncoupled Kinase Assay, reported in terms of specific activity (mg/mL). Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose. A total of five hexose substrates were tested in vitro, detailed in the table below. Based on these results, we were able to strengthen our initial hypothesis and continue characterization.<br /> <br /> [[Image:Specific_activity_table.png|400px|]]<br /> For further validation, we conducted an SDS analysis and provided below is the gel image. Indicated by the black box is our POI, around 34 kDa. Results were not as clear as anticipated, and in future studies, we would need to utilize different chromatography methods to yield higher quality protein concentrations and conduct a pre and post induction to visualize the purity of our protein.<br /> <br /> [[Image:FINAL_gel.png|250px|]]<br /> <br /> == Project Implications ==<br /> The goal of this project is to explore the techniques it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, including bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE. These techniques can be used to help characterize further putative kinases discovered in the future that do not have a defined function. This project is of importance because proteins are biomolecules responsible for as organisms' survival and understanding their unique function is essential for advances in modern medicine, scientific research, and agriculture.<br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> After various experiments and discussion, we have concluded that the novel protein 3R8E is a glucose kinase. <br /> <br /> - Authenticated and verified protein function using kinase characterization protocols<br /> <br /> - D-Glucose exhibited a specific activity of ___ ug/mL<br /> <br /> - Specific activity: D-Glucose &gt;&gt; D-Galactose &gt; D-Fructose &gt; Lactose &gt; D-Ribose<br /> <br /> - Protein 3r8e is a Glucose Kinase<br /> <br /> - Glucose is phosphorylated by our protein<br /> <br /> <br /> Future Direction:<br /> <br /> - Publish results for further application<br /> <br /> <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3550919 2022-04-26T03:23:34Z

<p>Michel Evertsen: </p> <hr /> <div>==Characterization of the 3r8e Protein, a Novel Gluco Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, and coupled kinase activity assays were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Future directions include repeating the above experiments and testing additional hexose substrates to further characterize the enzymatic activity of 3r8e. Overall, we have strong preliminary evidence that the 3r8e protein is a glucose kinase and future work will allow us to confirm this. <br /> == Introduction ==<br /> Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Besides the relationship with bacteria in soil, the role of this protein is still undetermined. As apart of a research project under the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) consortium, our group was tasked with characterizing and identifying the function of this protein to potentially provide further insight of the protein's relationship to the bacteria. Below is the general workflow of how we got our results. <br /> <br /> [[Image:Workflow1.png|500px|]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Alignment and highly conserved amino acids can be seen below.<br /> <br /> [[Image:3R8EDALIHMM.png|500px|]] [[Image:3R8EDali180.png|500px|]]<br /> These results along with supporting evidence from the other in silico tools allowed us to then conduct in silico docking experiments to further confirm our hypothesis. Below is a figure with the PyRx docking results of glucose and ATP with our protein. <br /> <br /> [[Image:PyRx1.png|500px|]]<br /> The -5.1 kcal/mol value shows that our protein of interest hypothesis of a glucose kinase is strong. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro. Because ATP is a commonality amongst sugar kinases, an &lt;scene name='90/904995/3r8ec_w_glc_and_atp/1'&gt;interactive structure&lt;/scene&gt; has been provided to represent glucose and ATP in the active site.<br /> == Experimental Results/Function ==<br /> <br /> Below are the results of our Uncoupled Kinase Assay. Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose.<br /> <br /> [[Image:KinaseAssayResults.png|500px|]]<br /> == Project Implications ==<br /> The goal of this project is to explore the methods it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, such as bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE Gel Electrophoresis. Proteins are biomolecules essential to organisms' survival and understanding how they work in result of their function is pivotal for advances in modern medicine, scientific research, and agriculture.<br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> - D-Glucose exhibited a specific activity of 4.22 ug/mL<br /> <br /> - Specific activity: D-Glucose &gt;&gt; D-Galactose &gt; D-Fructose &gt; Lactose &gt; D-Ribose<br /> <br /> - Protein 3r8e is a Glucose Kinase<br /> <br /> - Glucose is phosphorylated by our protein<br /> <br /> <br /> Future Direction:<br /> <br /> - Verify experimental results<br /> <br /> - Publish results for further application<br /> <br /> - Authenticate protein function using further kinase characterization protocols<br /> <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3550918 2022-04-26T03:22:53Z

<p>Michel Evertsen: </p> <hr /> <div>==Characterization of the 3r8e Protein, a Novel Gluco Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, and coupled kinase activity assays were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Future directions include repeating the above experiments and testing additional hexose substrates to further characterize the enzymatic activity of 3r8e. Overall, we have strong preliminary evidence that the 3r8e protein is a glucose kinase and future work will allow us to confirm this. <br /> == Introduction ==<br /> Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Besides the relationship with bacteria in soil, the role of this protein is still undetermined. As apart of a research project under the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) consortium, our group was tasked with characterizing and identifying the function of this protein to potentially provide further insight of the protein's relationship to the bacteria. Below is the general workflow of how we got our results. <br /> <br /> [[Image:Workflow1|500px|.png]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Alignment and highly conserved amino acids can be seen below.<br /> <br /> [[Image:3R8EDALIHMM.png|500px|]] [[Image:3R8EDali180.png|500px|]]<br /> These results along with supporting evidence from the other in silico tools allowed us to then conduct in silico docking experiments to further confirm our hypothesis. Below is a figure with the PyRx docking results of glucose and ATP with our protein. <br /> <br /> [[Image:PyRx1.png|500px|]]<br /> The -5.1 kcal/mol value shows that our protein of interest hypothesis of a glucose kinase is strong. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro. Because ATP is a commonality amongst sugar kinases, an &lt;scene name='90/904995/3r8ec_w_glc_and_atp/1'&gt;interactive structure&lt;/scene&gt; has been provided to represent glucose and ATP in the active site.<br /> == Experimental Results/Function ==<br /> <br /> Below are the results of our Uncoupled Kinase Assay. Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose.<br /> <br /> [[Image:KinaseAssayResults.png|500px|]]<br /> == Project Implications ==<br /> The goal of this project is to explore the methods it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, such as bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE Gel Electrophoresis. Proteins are biomolecules essential to organisms' survival and understanding how they work in result of their function is pivotal for advances in modern medicine, scientific research, and agriculture.<br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> - D-Glucose exhibited a specific activity of 4.22 ug/mL<br /> <br /> - Specific activity: D-Glucose &gt;&gt; D-Galactose &gt; D-Fructose &gt; Lactose &gt; D-Ribose<br /> <br /> - Protein 3r8e is a Glucose Kinase<br /> <br /> - Glucose is phosphorylated by our protein<br /> <br /> <br /> Future Direction:<br /> <br /> - Verify experimental results<br /> <br /> - Publish results for further application<br /> <br /> - Authenticate protein function using further kinase characterization protocols<br /> <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=File:Workflow1.png&diff=3550917 2022-04-26T03:21:52Z

<p>Michel Evertsen: </p> <hr /> <div></div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3550913 2022-04-26T03:14:34Z

<p>Michel Evertsen: </p> <hr /> <div>==Characterization of the 3r8e Protein, a Novel Gluco Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, and coupled kinase activity assays were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Future directions include repeating the above experiments and testing additional hexose substrates to further characterize the enzymatic activity of 3r8e. Overall, we have strong preliminary evidence that the 3r8e protein is a glucose kinase and future work will allow us to confirm this. <br /> == Introduction ==<br /> Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Besides the relationship with bacteria in soil, the role of this protein is still undetermined. As apart of a research project under the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) consortium, our group was tasked with characterizing and identifying the function of this protein to potentially provide further insight of the protein's relationship to the bacteria. Below is the general workflow of how we got our results. <br /> <br /> [[Image:Workflow.png ]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Alignment and highly conserved amino acids can be seen below.<br /> <br /> [[Image:3R8EDALIHMM.png|500px|]] [[Image:3R8EDali180.png|500px|]]<br /> These results along with supporting evidence from the other in silico tools allowed us to then conduct in silico docking experiments to further confirm our hypothesis. Below is a figure with the PyRx docking results of glucose and ATP with our protein. <br /> <br /> [[Image:PyRx1.png|500px|]]<br /> The -5.1 kcal/mol value shows that our protein of interest hypothesis of a glucose kinase is strong. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro. Because ATP is a commonality amongst sugar kinases, an &lt;scene name='90/904995/3r8ec_w_glc_and_atp/1'&gt;interactive structure&lt;/scene&gt; has been provided to represent glucose and ATP in the active site.<br /> == Experimental Results/Function ==<br /> <br /> Below are the results of our Uncoupled Kinase Assay. Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose.<br /> <br /> [[Image:KinaseAssayResults.png|500px|]]<br /> == Project Implications ==<br /> The goal of this project is to explore the methods it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, such as bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE Gel Electrophoresis. Proteins are biomolecules essential to organisms' survival and understanding how they work in result of their function is pivotal for advances in modern medicine, scientific research, and agriculture.<br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> - D-Glucose exhibited a specific activity of 4.22 ug/mL<br /> <br /> - Specific activity: D-Glucose &gt;&gt; D-Galactose &gt; D-Fructose &gt; Lactose &gt; D-Ribose<br /> <br /> - Protein 3r8e is a Glucose Kinase<br /> <br /> - Glucose is phosphorylated by our protein<br /> <br /> <br /> Future Direction:<br /> <br /> - Verify experimental results<br /> <br /> - Publish results for further application<br /> <br /> - Authenticate protein function using further kinase characterization protocols<br /> <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=File:3R8EDali180.png&diff=3550912 2022-04-26T03:13:36Z

<p>Michel Evertsen: </p> <hr /> <div></div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3550911 2022-04-26T03:11:04Z

<p>Michel Evertsen: </p> <hr /> <div>==Characterization of the 3r8e Protein, a Novel Gluco Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, and coupled kinase activity assays were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Future directions include repeating the above experiments and testing additional hexose substrates to further characterize the enzymatic activity of 3r8e. Overall, we have strong preliminary evidence that the 3r8e protein is a glucose kinase and future work will allow us to confirm this. <br /> == Introduction ==<br /> Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Besides the relationship with bacteria in soil, the role of this protein is still undetermined. As apart of a research project under the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) consortium, our group was tasked with characterizing and identifying the function of this protein to potentially provide further insight of the protein's relationship to the bacteria. Below is the general workflow of how we got our results. <br /> <br /> [[Image:Workflow.png ]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Alignment and highly conserved amino acids can be seen below.<br /> <br /> [[Image:3R8EDALIHMM.png|500px|]]<br /> These results along with supporting evidence from the other in silico tools allowed us to then conduct in silico docking experiments to further confirm our hypothesis. Below is a figure with the PyRx docking results of glucose and ATP with our protein. <br /> <br /> [[Image:PyRx1.png|500px|]]<br /> The -5.1 kcal/mol value shows that our protein of interest hypothesis of a glucose kinase is strong. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro. Because ATP is a commonality amongst sugar kinases, an &lt;scene name='90/904995/3r8ec_w_glc_and_atp/1'&gt;interactive structure&lt;/scene&gt; has been provided to represent glucose and ATP in the active site.<br /> == Experimental Results/Function ==<br /> <br /> Below are the results of our Uncoupled Kinase Assay. Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose.<br /> <br /> [[Image:KinaseAssayResults.png|500px|]]<br /> == Project Implications ==<br /> The goal of this project is to explore the methods it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, such as bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE Gel Electrophoresis. Proteins are biomolecules essential to organisms' survival and understanding how they work in result of their function is pivotal for advances in modern medicine, scientific research, and agriculture.<br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> - D-Glucose exhibited a specific activity of 4.22 ug/mL<br /> <br /> - Specific activity: D-Glucose &gt;&gt; D-Galactose &gt; D-Fructose &gt; Lactose &gt; D-Ribose<br /> <br /> - Protein 3r8e is a Glucose Kinase<br /> <br /> - Glucose is phosphorylated by our protein<br /> <br /> <br /> Future Direction:<br /> <br /> - Verify experimental results<br /> <br /> - Publish results for further application<br /> <br /> - Authenticate protein function using further kinase characterization protocols<br /> <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=File:3R8EDALIHMM.png&diff=3550910 2022-04-26T03:10:32Z

<p>Michel Evertsen: </p> <hr /> <div></div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3550907 2022-04-26T03:05:34Z

<p>Michel Evertsen: </p> <hr /> <div>==Characterization of the 3r8e Protein, a Novel Gluco Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, and coupled kinase activity assays were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Future directions include repeating the above experiments and testing additional hexose substrates to further characterize the enzymatic activity of 3r8e. Overall, we have strong preliminary evidence that the 3r8e protein is a glucose kinase and future work will allow us to confirm this. <br /> == Introduction ==<br /> Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Besides the relationship with bacteria in soil, the role of this protein is still undetermined. As apart of a research project under the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) consortium, our group was tasked with characterizing and identifying the function of this protein to potentially provide further insight of the protein's relationship to the bacteria. Below is the general workflow of how we got our results. <br /> <br /> [[Image:Workflow.png ]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Alignment and highly conserved amino acids can be seen below.<br /> <br /> [[Image:3R8EDALI.png|500px|]]<br /> These results along with supporting evidence from the other in silico tools allowed us to then conduct in silico docking experiments to further confirm our hypothesis. Below is a figure with the PyRx docking results of glucose and ATP with our protein. <br /> <br /> [[Image:PyRx1.png|500px|]]<br /> The -5.1 kcal/mol value shows that our protein of interest hypothesis of a glucose kinase is strong. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro. Because ATP is a commonality amongst sugar kinases, an &lt;scene name='90/904995/3r8ec_w_glc_and_atp/1'&gt;interactive structure&lt;/scene&gt; has been provided to represent glucose and ATP in the active site.<br /> == Experimental Results/Function ==<br /> <br /> Below are the results of our Uncoupled Kinase Assay. Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose.<br /> <br /> [[Image:KinaseAssayResults.png|500px|]]<br /> == Project Implications ==<br /> The goal of this project is to explore the methods it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, such as bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE Gel Electrophoresis. Proteins are biomolecules essential to organisms' survival and understanding how they work in result of their function is pivotal for advances in modern medicine, scientific research, and agriculture.<br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> - D-Glucose exhibited a specific activity of 4.22 ug/mL<br /> <br /> - Specific activity: D-Glucose &gt;&gt; D-Galactose &gt; D-Fructose &gt; Lactose &gt; D-Ribose<br /> <br /> - Protein 3r8e is a Glucose Kinase<br /> <br /> - Glucose is phosphorylated by our protein<br /> <br /> <br /> Future Direction:<br /> <br /> - Verify experimental results<br /> <br /> - Publish results for further application<br /> <br /> - Authenticate protein function using further kinase characterization protocols<br /> <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3550906 2022-04-26T03:04:23Z

<p>Michel Evertsen: </p> <hr /> <div>==Characterization of the 3r8e Protein, a Novel Gluco Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, and coupled kinase activity assays were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Future directions include repeating the above experiments and testing additional hexose substrates to further characterize the enzymatic activity of 3r8e. Overall, we have strong preliminary evidence that the 3r8e protein is a glucose kinase and future work will allow us to confirm this. <br /> == Introduction ==<br /> Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Besides the relationship with bacteria in soil, the role of this protein is still undetermined. As apart of a research project under the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) consortium, our group was tasked with characterizing and identifying the function of this protein to potentially provide further insight of the protein's relationship to the bacteria. Below is the general workflow of how we got our results. <br /> <br /> [[Image:Workflow.png ]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Alignment and highly conserved amino acids can be seen below.<br /> <br /> [[Image:3R8EDALI.png|300px|left|thumb|]]<br /> These results along with supporting evidence from the other in silico tools allowed us to then conduct in silico docking experiments to further confirm our hypothesis. Below is a figure with the PyRx docking results of glucose and ATP with our protein. <br /> <br /> [[Image:PyRx1.png|300px|left|thumb|]]<br /> The -5.1 kcal/mol value shows that our protein of interest hypothesis of a glucose kinase is strong. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro. Because ATP is a commonality amongst sugar kinases, an &lt;scene name='90/904995/3r8ec_w_glc_and_atp/1'&gt;interactive structure&lt;/scene&gt; has been provided to represent glucose and ATP in the active site.<br /> == Experimental Results/Function ==<br /> <br /> Below are the results of our Uncoupled Kinase Assay. Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose.<br /> <br /> [[Image:KinaseAssayResults.png|300px|left|thumb|]]<br /> == Project Implications ==<br /> The goal of this project is to explore the methods it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, such as bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE Gel Electrophoresis. Proteins are biomolecules essential to organisms' survival and understanding how they work in result of their function is pivotal for advances in modern medicine, scientific research, and agriculture.<br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> - D-Glucose exhibited a specific activity of 4.22 ug/mL<br /> <br /> - Specific activity: D-Glucose &gt;&gt; D-Galactose &gt; D-Fructose &gt; Lactose &gt; D-Ribose<br /> <br /> - Protein 3r8e is a Glucose Kinase<br /> <br /> - Glucose is phosphorylated by our protein<br /> <br /> <br /> Future Direction:<br /> <br /> - Verify experimental results<br /> <br /> - Publish results for further application<br /> <br /> - Authenticate protein function using further kinase characterization protocols<br /> <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=File:3R8EDALI.png&diff=3550905 2022-04-26T03:02:18Z

<p>Michel Evertsen: </p> <hr /> <div></div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3547686 2022-04-21T01:16:13Z

<p>Michel Evertsen: </p> <hr /> <div>==Characterization of the 3r8e Protein, a Novel Gluco Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, and coupled kinase activity assays were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Future directions include repeating the above experiments and testing additional hexose substrates to further characterize the enzymatic activity of 3r8e. Overall, we have strong preliminary evidence that the 3r8e protein is a glucose kinase and future work will allow us to confirm this.<br /> == Introduction ==<br /> Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Extracted from and being a part of a bacteria that is found in soil, no one knows what the role of this specific protein plays in relation to the bacteria. Apart of a research project, our group set out to solve the function of this protein to then potentially provide further insight of the protein's relationship to the bacteria. Below is the general workflow of how we got our results. <br /> <br /> [[Image:Workflow.png ]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Alignment and highly conserved amino acids can be seen below.<br /> <br /> [[Image:DALI.png]]<br /> These results along with supporting evidence from the other in silico tools allowed us to then conduct in silico docking experiments to further confirm our hypothesis. Below is a figure with the PyRx docking results of glucose and ATP with our protein. <br /> <br /> [[Image:PyRx1.png]]<br /> The -5.1 kcal/mol value shows that our protein of interest hypothesis of a glucose kinase is strong. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro.<br /> == Experimental Results/Function ==<br /> <br /> Below are the results of our Uncoupled Kinase Assay<br /> <br /> [[Image:KinaseAssayResults.png]]<br /> == Project Implications ==<br /> <br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> - D-Glucose exhibited a specific activity of 4.22 ug/mL<br /> <br /> - Specific activity: D-Glucose &gt;&gt; D-Galactose &gt; D-Fructose &gt; Lactose &gt; D-Ribose<br /> <br /> - Protein 3r8e is a Glucose Kinase<br /> <br /> - Glucose is phosphorylated by our protein<br /> <br /> <br /> Future Direction:<br /> <br /> - Verify experimental results<br /> <br /> - Publish results for further application<br /> <br /> - Authenticate protein function using further kinase characterization protocols<br /> <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3547685 2022-04-21T01:15:49Z

<p>Michel Evertsen: </p> <hr /> <div>==Characterization of the 3r8e Protein, a Novel Gluco Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, and coupled kinase activity assays were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Future directions include repeating the above experiments and testing additional hexose substrates to further characterize the enzymatic activity of 3r8e. Overall, we have strong preliminary evidence that the 3r8e protein is a glucose kinase and future work will allow us to confirm this.<br /> == Introduction ==<br /> Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Extracted from and being a part of a bacteria that is found in soil, no one knows what the role of this specific protein plays in relation to the bacteria. Apart of a research project, our group set out to solve the function of this protein to then potentially provide further insight of the protein's relationship to the bacteria. Below is the general workflow of how we got our results. <br /> <br /> [[Image:Workflow.png ]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Alignment and highly conserved amino acids can be seen below.<br /> <br /> [[Image:DALI.png]]<br /> These results along with supporting evidence from the other in silico tools allowed us to then conduct in silico docking experiments to further confirm our hypothesis. Below is a figure with the PyRx docking results of glucose and ATP with our protein. <br /> <br /> [[Image:PyRx1.png]]<br /> The -5.1 kcal/mol value shows that our protein of interest hypothesis of a glucose kinase is strong. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro.<br /> == Experimental Results/Function ==<br /> <br /> Below are the results of our Uncoupled Kinase Assay<br /> [[Image:KinaseAssayResults.png]]<br /> == Project Implications ==<br /> <br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> - D-Glucose exhibited a specific activity of 4.22 ug/mL<br /> <br /> - Specific activity: D-Glucose &gt;&gt; D-Galactose &gt; D-Fructose &gt; Lactose &gt; D-Ribose<br /> <br /> - Protein 3r8e is a Glucose Kinase<br /> <br /> - Glucose is phosphorylated by our protein<br /> <br /> <br /> Future Direction:<br /> <br /> - Verify experimental results<br /> <br /> - Publish results for further application<br /> <br /> - Authenticate protein function using further kinase characterization protocols<br /> <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3547684 2022-04-21T01:14:19Z

<p>Michel Evertsen: </p> <hr /> <div>==Characterization of the 3r8e Protein, a Novel Gluco Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, and coupled kinase activity assays were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Future directions include repeating the above experiments and testing additional hexose substrates to further characterize the enzymatic activity of 3r8e. Overall, we have strong preliminary evidence that the 3r8e protein is a glucose kinase and future work will allow us to confirm this.<br /> == Introduction ==<br /> Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Extracted from and being a part of a bacteria that is found in soil, no one knows what the role of this specific protein plays in relation to the bacteria. Apart of a research project, our group set out to solve the function of this protein to then potentially provide further insight of the protein's relationship to the bacteria. Below is the general workflow of how we got our results. <br /> <br /> [[Image:Workflow.png ]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Alignment and highly conserved amino acids can be seen below.<br /> <br /> [[Image:DALI.png]]<br /> These results along with supporting evidence from the other in silico tools allowed us to then conduct in silico docking experiments to further confirm our hypothesis. Below is a figure with the PyRx docking results of glucose and ATP with our protein. <br /> <br /> [[Image:PyRx1.png]]<br /> The -5.1 kcal/mol value shows that our protein of interest hypothesis of a glucose kinase is strong. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro.<br /> == Experimental Results/Function ==<br /> <br /> Below are the results of our Uncoupled Kinase Assay<br /> <br /> [[Image:KinaseAssayResults.png]]<br /> == Project Implications ==<br /> <br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> - D-Glucose exhibited a specific activity of 4.22 ug/mL<br /> <br /> - Specific activity: D-Glucose &gt;&gt; D-Galactose &gt; D-Fructose &gt; Lactose &gt; D-Ribose<br /> <br /> - Protein 3r8e is a Glucose Kinase<br /> <br /> - Glucose is phosphorylated by our protein<br /> <br /> <br /> Future Direction:<br /> <br /> - Verify experimental results<br /> <br /> - Publish results for further application<br /> <br /> - Authenticate protein function using further kinase characterization protocols<br /> <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3547683 2022-04-21T01:13:58Z

<p>Michel Evertsen: </p> <hr /> <div>==Characterization of the 3r8e Protein, a Novel Gluco Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, and coupled kinase activity assays were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Future directions include repeating the above experiments and testing additional hexose substrates to further characterize the enzymatic activity of 3r8e. Overall, we have strong preliminary evidence that the 3r8e protein is a glucose kinase and future work will allow us to confirm this.<br /> == Introduction ==<br /> Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Extracted from and being a part of a bacteria that is found in soil, no one knows what the role of this specific protein plays in relation to the bacteria. Apart of a research project, our group set out to solve the function of this protein to then potentially provide further insight of the protein's relationship to the bacteria. Below is the general workflow of how we got our results. <br /> <br /> [[Image:Workflow.png ]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Alignment and highly conserved amino acids can be seen below.<br /> <br /> [[Image:DALI.png]]<br /> These results along with supporting evidence from the other in silico tools allowed us to then conduct in silico docking experiments to further confirm our hypothesis. Below is a figure with the PyRx docking results of glucose and ATP with our protein. <br /> <br /> [[Image:PyRx1.png]]<br /> The -5.1 kcal/mol value shows that our protein of interest hypothesis of a glucose kinase is strong. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro.<br /> == Experimental Results/Function ==<br /> <br /> Below are the results of our Uncoupled Kinase Assay<br /> [[Image:KinaseAssayResults.png]]<br /> == Project Implications ==<br /> <br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> - D-Glucose exhibited a specific activity of 4.22 ug/mL<br /> <br /> - Specific activity: D-Glucose &gt;&gt; D-Galactose &gt; D-Fructose &gt; Lactose &gt; D-Ribose<br /> <br /> - Protein 3r8e is a Glucose Kinase<br /> <br /> - Glucose is phosphorylated by our protein<br /> <br /> <br /> Future Direction:<br /> <br /> - Verify experimental results<br /> <br /> - Publish results for further application<br /> <br /> - Authenticate protein function using further kinase characterization protocols<br /> <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=File:KinaseAssayResults.png&diff=3547682 2022-04-21T01:12:31Z

<p>Michel Evertsen: </p> <hr /> <div></div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3547675 2022-04-21T01:01:15Z

<p>Michel Evertsen: </p> <hr /> <div>==Characterization of the 3r8e Protein, a Novel Gluco Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, and coupled kinase activity assays were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Future directions include repeating the above experiments and testing additional hexose substrates to further characterize the enzymatic activity of 3r8e. Overall, we have strong preliminary evidence that the 3r8e protein is a glucose kinase and future work will allow us to confirm this.<br /> == Introduction ==<br /> Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Extracted from and being a part of a bacteria that is found in soil, no one knows what the role of this specific protein plays in relation to the bacteria. Apart of a research project, our group set out to solve the function of this protein to then potentially provide further insight of the protein's relationship to the bacteria. Below is the general workflow of how we got our results. <br /> <br /> [[Image:Workflow.png ]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Alignment and highly conserved amino acids can be seen below.<br /> <br /> [[Image:DALI.png]]<br /> These results along with supporting evidence from the other in silico tools allowed us to then conduct in silico docking experiments to further confirm our hypothesis. Below is a figure with the PyRx docking results of glucose and ATP with our protein. <br /> <br /> [[Image:PyRx1.png]]<br /> The -5.1 kcal/mol value shows that our protein of interest hypothesis of a glucose kinase is strong. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro.<br /> == Experimental Results/Function ==<br /> <br /> == Project Implications ==<br /> <br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> - D-Glucose exhibited a specific activity of 4.22 ug/mL<br /> <br /> - Specific activity: D-Glucose &gt;&gt; D-Galactose &gt; D-Fructose &gt; Lactose &gt; D-Ribose<br /> <br /> - Protein 3r8e is a Glucose Kinase<br /> <br /> - Glucose is phosphorylated by our protein<br /> <br /> <br /> Future Direction:<br /> <br /> - Verify experimental results<br /> <br /> - Publish results for further application<br /> <br /> - Authenticate protein function using further kinase characterization protocols<br /> <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=File:PyRx1.png&diff=3547674 2022-04-21T01:00:23Z

<p>Michel Evertsen: </p> <hr /> <div></div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3547670 2022-04-21T00:58:00Z

<p>Michel Evertsen: </p> <hr /> <div>==Characterization of the 3r8e Protein, a Novel Gluco Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, and coupled kinase activity assays were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Future directions include repeating the above experiments and testing additional hexose substrates to further characterize the enzymatic activity of 3r8e. Overall, we have strong preliminary evidence that the 3r8e protein is a glucose kinase and future work will allow us to confirm this.<br /> == Introduction ==<br /> Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Extracted from and being a part of a bacteria that is found in soil, no one knows what the role of this specific protein plays in relation to the bacteria. Apart of a research project, our group set out to solve the function of this protein to then potentially provide further insight of the protein's relationship to the bacteria. Below is the general workflow of how we got our results. <br /> <br /> [[Image:Workflow.png ]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Alignment and highly conserved amino acids can be seen below.<br /> <br /> [[Image:DALI.png]]<br /> These results along with supporting evidence from the other in silico tools allowed us to then conduct in silico docking experiments to further confirm our hypothesis. Below is a figure with the PyRx docking results of glucose and ATP with our protein. <br /> <br /> [[Image:PyRx.png]]<br /> The -5.1 kcal/mol value shows that our protein of interest hypothesis of a glucose kinase is strong. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro.<br /> == Experimental Results/Function ==<br /> <br /> == Project Implications ==<br /> <br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> - D-Glucose exhibited a specific activity of 4.22 ug/mL<br /> <br /> - Specific activity: D-Glucose &gt;&gt; D-Galactose &gt; D-Fructose &gt; Lactose &gt; D-Ribose<br /> <br /> - Protein 3r8e is a Glucose Kinase<br /> <br /> - Glucose is phosphorylated by our protein<br /> <br /> <br /> Future Direction:<br /> <br /> - Verify experimental results<br /> <br /> - Publish results for further application<br /> <br /> - Authenticate protein function using further kinase characterization protocols<br /> <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 2. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 5. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3547658 2022-04-21T00:33:10Z

<p>Michel Evertsen: </p> <hr /> <div>==Characterization of the 3r8e Protein, a Novel Gluco Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, and coupled kinase activity assays were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Future directions include repeating the above experiments and testing additional hexose substrates to further characterize the enzymatic activity of 3r8e. Overall, we have strong preliminary evidence that the 3r8e protein is a glucose kinase and future work will allow us to confirm this.<br /> == Introduction ==<br /> Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Extracted from and being a part of a bacteria that is found in soil, no one knows what the role of this specific protein plays in relation to the bacteria. Apart of a research project, our group set out to solve the function of this protein to then potentially provide further insight of the protein's relationship to the bacteria. Below is the general workflow of how we got our results. <br /> <br /> [[Image:Workflow.png ]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Alignment and highly conserved amino acids can be seen below.<br /> <br /> [[Image:DALI.png]]<br /> These results along with supporting evidence from the other in silico tools allowed us to then conduct in silico docking experiments to further confirm our hypothesis. Below is a figure with the PyRx docking results of glucose and ATP with our protein. <br /> <br /> [[Image:PyRx.png]]<br /> The -5.1 kcal/mol value shows that our protein of interest hypothesis of a glucose kinase is strong. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro.<br /> == Experimental Results/Function ==<br /> <br /> == Project Implications ==<br /> <br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> - D-Glucose exhibited a specific activity of 4.22 ug/mL<br /> <br /> - Specific activity: D-Glucose &gt;&gt; D-Galactose &gt; D-Fructose &gt; Lactose &gt; D-Ribose<br /> <br /> - Protein 3r8e is a Glucose Kinase<br /> <br /> - Glucose is phosphorylated by our protein<br /> <br /> <br /> Future Direction:<br /> <br /> - Verify experimental results<br /> <br /> - Publish results for further application<br /> <br /> - Authenticate protein function using further kinase characterization protocols<br /> <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 3. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 4. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 5. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 6. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 7. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 8. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3547657 2022-04-21T00:32:36Z

<p>Michel Evertsen: </p> <hr /> <div>==Characterization of the 3r8e Protein, a Novel Gluco Kinase==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> This is a default text for your page '''BASIL2022GV3R8E'''. Click above on '''edit this page''' to modify. Be careful with the &amp;lt; and &amp;gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia &lt;ref&gt;DOI 10.1002/ijch.201300024&lt;/ref&gt; or to the article describing Jmol &lt;ref&gt;PMID:21638687&lt;/ref&gt; to the rescue.<br /> <br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, and coupled kinase activity assays were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Future directions include repeating the above experiments and testing additional hexose substrates to further characterize the enzymatic activity of 3r8e. Overall, we have strong preliminary evidence that the 3r8e protein is a glucose kinase and future work will allow us to confirm this.<br /> == Introduction ==<br /> Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Extracted from and being a part of a bacteria that is found in soil, no one knows what the role of this specific protein plays in relation to the bacteria. Apart of a research project, our group set out to solve the function of this protein to then potentially provide further insight of the protein's relationship to the bacteria. Below is the general workflow of how we got our results. <br /> <br /> [[Image:Workflow.png ]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Alignment and highly conserved amino acids can be seen below.<br /> <br /> [[Image:DALI.png]]<br /> These results along with supporting evidence from the other in silico tools allowed us to then conduct in silico docking experiments to further confirm our hypothesis. Below is a figure with the PyRx docking results of glucose and ATP with our protein. <br /> <br /> [[Image:PyRx.png]]<br /> The -5.1 kcal/mol value shows that our protein of interest hypothesis of a glucose kinase is strong. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro.<br /> == Experimental Results/Function ==<br /> <br /> == Project Implications ==<br /> <br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> - D-Glucose exhibited a specific activity of 4.22 ug/mL<br /> <br /> - Specific activity: D-Glucose &gt;&gt; D-Galactose &gt; D-Fructose &gt; Lactose &gt; D-Ribose<br /> <br /> - Protein 3r8e is a Glucose Kinase<br /> <br /> - Glucose is phosphorylated by our protein<br /> <br /> <br /> Future Direction:<br /> <br /> - Verify experimental results<br /> <br /> - Publish results for further application<br /> <br /> - Authenticate protein function using further kinase characterization protocols<br /> <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 3. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 4. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 5. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 6. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 7. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 8. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3547654 2022-04-21T00:31:05Z

<p>Michel Evertsen: </p> <hr /> <div>==Your Heading Here (maybe something like 'Structure')==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> This is a default text for your page '''BASIL2022GV3R8E'''. Click above on '''edit this page''' to modify. Be careful with the &amp;lt; and &amp;gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia &lt;ref&gt;DOI 10.1002/ijch.201300024&lt;/ref&gt; or to the article describing Jmol &lt;ref&gt;PMID:21638687&lt;/ref&gt; to the rescue.<br /> <br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, and coupled kinase activity assays were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Future directions include repeating the above experiments and testing additional hexose substrates to further characterize the enzymatic activity of 3r8e. Overall, we have strong preliminary evidence that the 3r8e protein is a glucose kinase and future work will allow us to confirm this.<br /> == Introduction ==<br /> Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Extracted from and being a part of a bacteria that is found in soil, no one knows what the role of this specific protein plays in relation to the bacteria. Apart of a research project, our group set out to solve the function of this protein to then potentially provide further insight of the protein's relationship to the bacteria. Below is the general workflow of how we got our results. <br /> <br /> [[Image:Workflow.png ]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Alignment and highly conserved amino acids can be seen below.<br /> <br /> [[Image:DALI.png]]<br /> These results along with supporting evidence from the other in silico tools allowed us to then conduct in silico docking experiments to further confirm our hypothesis. Below is a figure with the PyRx docking results of glucose and ATP with our protein. <br /> <br /> [[Image:PyRx.png]]<br /> The -5.1 kcal/mol value shows that our protein of interest hypothesis of a glucose kinase is strong. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro.<br /> == Experimental Results/Function ==<br /> <br /> == Project Implications ==<br /> <br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> <br /> - D-Glucose exhibited a specific activity of 4.22 ug/mL<br /> <br /> - Specific activity: D-Glucose &gt;&gt; D-Galactose &gt; D-Fructose &gt; Lactose &gt; D-Ribose<br /> <br /> - Protein 3r8e is a Glucose Kinase<br /> <br /> - Glucose is phosphorylated by our protein<br /> <br /> <br /> Future Direction:<br /> <br /> - Verify experimental results<br /> <br /> - Publish results for further application<br /> <br /> - Authenticate protein function using further kinase characterization protocols<br /> <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 3. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 4. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 5. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 6. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 7. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 8. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3547652 2022-04-21T00:30:01Z

<p>Michel Evertsen: </p> <hr /> <div>==Your Heading Here (maybe something like 'Structure')==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> This is a default text for your page '''BASIL2022GV3R8E'''. Click above on '''edit this page''' to modify. Be careful with the &amp;lt; and &amp;gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia &lt;ref&gt;DOI 10.1002/ijch.201300024&lt;/ref&gt; or to the article describing Jmol &lt;ref&gt;PMID:21638687&lt;/ref&gt; to the rescue.<br /> <br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, and coupled kinase activity assays were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Future directions include repeating the above experiments and testing additional hexose substrates to further characterize the enzymatic activity of 3r8e. Overall, we have strong preliminary evidence that the 3r8e protein is a glucose kinase and future work will allow us to confirm this.<br /> == Introduction ==<br /> Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Extracted from and being a part of a bacteria that is found in soil, no one knows what the role of this specific protein plays in relation to the bacteria. Apart of a research project, our group set out to solve the function of this protein to then potentially provide further insight of the protein's relationship to the bacteria. Below is the general workflow of how we got our results. <br /> <br /> [[Image:Workflow.png ]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Alignment and highly conserved amino acids can be seen below.<br /> <br /> [[Image:DALI.png]]<br /> These results along with supporting evidence from the other in silico tools allowed us to then conduct in silico docking experiments to further confirm our hypothesis. Below is a figure with the PyRx docking results of glucose and ATP with our protein. <br /> <br /> [[Image:PyRx.png]]<br /> The -5.1 kcal/mol value shows that our protein of interest hypothesis of a glucose kinase is strong. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro.<br /> == Experimental Results/Function ==<br /> <br /> == Project Implications ==<br /> <br /> == Conclusions/Future Direction ==<br /> Conclusions:<br /> - D-Glucose exhibited a specific activity of 4.22 ug/mL<br /> - Specific activity: D-Glucose &gt;&gt; D-Galactose &gt; D-Fructose &gt; Lactose &gt; D-Ribose<br /> - Protein 3r8e is a Glucose Kinase<br /> - Glucose is phosphorylated by our protein<br /> <br /> Future Direction:<br /> - Verify experimental results<br /> - Publish results for further application<br /> - Authenticate protein function using further kinase characterization protocols<br /> <br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 3. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 4. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 5. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 6. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 7. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 8. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3547650 2022-04-21T00:26:42Z

<p>Michel Evertsen: </p> <hr /> <div>==Your Heading Here (maybe something like 'Structure')==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> This is a default text for your page '''BASIL2022GV3R8E'''. Click above on '''edit this page''' to modify. Be careful with the &amp;lt; and &amp;gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia &lt;ref&gt;DOI 10.1002/ijch.201300024&lt;/ref&gt; or to the article describing Jmol &lt;ref&gt;PMID:21638687&lt;/ref&gt; to the rescue.<br /> <br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, and coupled kinase activity assays were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Future directions include repeating the above experiments and testing additional hexose substrates to further characterize the enzymatic activity of 3r8e. Overall, we have strong preliminary evidence that the 3r8e protein is a glucose kinase and future work will allow us to confirm this.<br /> == Introduction ==<br /> Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Extracted from and being a part of a bacteria that is found in soil, no one knows what the role of this specific protein plays in relation to the bacteria. Apart of a research project, our group set out to solve the function of this protein to then potentially provide further insight of the protein's relationship to the bacteria. Below is the general workflow of how we got our results. <br /> <br /> [[Image:Workflow.png ]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Alignment and highly conserved amino acids can be seen below.<br /> <br /> [[Image:DALI.png]]<br /> These results along with supporting evidence from the other in silico tools allowed us to then conduct in silico docking experiments to further confirm our hypothesis. Below is a figure with the PyRx docking results of glucose and ATP with our protein. <br /> <br /> [[Image:PyRx.png]]<br /> The -5.1 kcal/mol value shows that our protein of interest hypothesis of a glucose kinase is strong. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro.<br /> == Experimental Results/Function ==<br /> <br /> == Project Implications ==<br /> <br /> == Conclusions/Future Direction ==<br /> <br /> This is a sample scene created with SAT to &lt;scene name=&quot;/12/3456/Sample/1&quot;&gt;color&lt;/scene&gt; by Group, and another to make &lt;scene name=&quot;/12/3456/Sample/2&quot;&gt;a transparent representation&lt;/scene&gt; of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 3. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 4. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 5. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 6. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 7. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 8. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3547649 2022-04-21T00:26:18Z

<p>Michel Evertsen: </p> <hr /> <div>==Your Heading Here (maybe something like 'Structure')==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> This is a default text for your page '''BASIL2022GV3R8E'''. Click above on '''edit this page''' to modify. Be careful with the &amp;lt; and &amp;gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia &lt;ref&gt;DOI 10.1002/ijch.201300024&lt;/ref&gt; or to the article describing Jmol &lt;ref&gt;PMID:21638687&lt;/ref&gt; to the rescue.<br /> <br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, and coupled kinase activity assays were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Future directions include repeating the above experiments and testing additional hexose substrates to further characterize the enzymatic activity of 3r8e. Overall, we have strong preliminary evidence that the 3r8e protein is a glucose kinase and future work will allow us to confirm this.<br /> == Introduction ==<br /> Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Extracted from and being a part of a bacteria that is found in soil, no one knows what the role of this specific protein plays in relation to the bacteria. Apart of a research project, our group set out to solve the function of this protein to then potentially provide further insight of the protein's relationship to the bacteria. Below is the general workflow of how we got our results. <br /> <br /> [[Image:Workflow.png ]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Alignment and highly conserved amino acids can be seen below. <br /> [[Image:DALI.png]]<br /> These results along with supporting evidence from the other in silico tools allowed us to then conduct in silico docking experiments to further confirm our hypothesis. Below is a figure with the PyRx docking results of glucose and ATP with our protein. <br /> [[Image:PyRx.png]]<br /> The -5.1 kcal/mol value shows that our protein of interest hypothesis of a glucose kinase is strong. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro.<br /> == Experimental Results/Function ==<br /> <br /> == Project Implications ==<br /> <br /> == Conclusions/Future Direction ==<br /> <br /> This is a sample scene created with SAT to &lt;scene name=&quot;/12/3456/Sample/1&quot;&gt;color&lt;/scene&gt; by Group, and another to make &lt;scene name=&quot;/12/3456/Sample/2&quot;&gt;a transparent representation&lt;/scene&gt; of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 3. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 4. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 5. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 6. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 7. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 8. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=File:PyRx.png&diff=3547648 2022-04-21T00:25:24Z

<p>Michel Evertsen: </p> <hr /> <div></div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3547647 2022-04-21T00:23:34Z

<p>Michel Evertsen: </p> <hr /> <div>==Your Heading Here (maybe something like 'Structure')==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> This is a default text for your page '''BASIL2022GV3R8E'''. Click above on '''edit this page''' to modify. Be careful with the &amp;lt; and &amp;gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia &lt;ref&gt;DOI 10.1002/ijch.201300024&lt;/ref&gt; or to the article describing Jmol &lt;ref&gt;PMID:21638687&lt;/ref&gt; to the rescue.<br /> <br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, and coupled kinase activity assays were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Future directions include repeating the above experiments and testing additional hexose substrates to further characterize the enzymatic activity of 3r8e. Overall, we have strong preliminary evidence that the 3r8e protein is a glucose kinase and future work will allow us to confirm this.<br /> == Introduction ==<br /> Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Extracted from and being a part of a bacteria that is found in soil, no one knows what the role of this specific protein plays in relation to the bacteria. Apart of a research project, our group set out to solve the function of this protein to then potentially provide further insight of the protein's relationship to the bacteria. Below is the general workflow of how we got our results. <br /> <br /> [[Image:Workflow.png ]]<br /> == Methods ==<br /> As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Alignment and highly conserved amino acids can be seen below. <br /> [[Image:DALI.png]]<br /> These results along with supporting evidence from the other in silico tools allowed us to then conduct in silico docking experiments to further confirm our hypothesis. Below is a figure with the PyRx docking results of glucose and ATP with our protein. <br /> <br /> The -5.1 kcal/mol value shows that our protein of interest hypothesis of a glucose kinase is strong. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro.<br /> == Experimental Results/Function ==<br /> <br /> == Project Implications ==<br /> <br /> == Conclusions/Future Direction ==<br /> <br /> This is a sample scene created with SAT to &lt;scene name=&quot;/12/3456/Sample/1&quot;&gt;color&lt;/scene&gt; by Group, and another to make &lt;scene name=&quot;/12/3456/Sample/2&quot;&gt;a transparent representation&lt;/scene&gt; of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 3. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 4. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 5. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 6. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 7. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 8. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen https://proteopedia.org/index.php?title=File:DALI.png&diff=3547645 2022-04-21T00:21:45Z

<p>Michel Evertsen: uploaded a new version of &quot;Image:DALI.png&quot;</p> <hr /> <div> 3hdt aligned with PDB IDs: 7l4a and 1kdo</div>

Michel Evertsen https://proteopedia.org/index.php?title=BASIL2022GV3R8E&diff=3547609 2022-04-20T22:39:07Z

<p>Michel Evertsen: </p> <hr /> <div>==Your Heading Here (maybe something like 'Structure')==<br /> &lt;StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''&gt;<br /> This is a default text for your page '''BASIL2022GV3R8E'''. Click above on '''edit this page''' to modify. Be careful with the &amp;lt; and &amp;gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia &lt;ref&gt;DOI 10.1002/ijch.201300024&lt;/ref&gt; or to the article describing Jmol &lt;ref&gt;PMID:21638687&lt;/ref&gt; to the rescue.<br /> <br /> == Abstract ==<br /> The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, and coupled kinase activity assays were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Future directions include repeating the above experiments and testing additional hexose substrates to further characterize the enzymatic activity of 3r8e. Overall, we have strong preliminary evidence that the 3r8e protein is a glucose kinase and future work will allow us to confirm this.<br /> == Introduction ==<br /> Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Extracted from and being a part of a bacteria that is found in soil, no one knows what the role of this specific protein plays in relation to the bacteria. Apart of a research project, our group set out to solve the function of this protein to then potentially provide further insight of the protein's relationship to the bacteria. Below is the general workflow of how we got our results. <br /> <br /> [[Image:Workflow.png ]]<br /> == Methods ==<br /> <br /> == Experimental Results/Function ==<br /> <br /> == Project Implications ==<br /> <br /> == Conclusions/Future Direction ==<br /> <br /> This is a sample scene created with SAT to &lt;scene name=&quot;/12/3456/Sample/1&quot;&gt;color&lt;/scene&gt; by Group, and another to make &lt;scene name=&quot;/12/3456/Sample/2&quot;&gt;a transparent representation&lt;/scene&gt; of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> &lt;/StructureSection&gt;<br /> == References ==<br /> &lt;references/&gt;<br /> 3. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)<br /> <br /> 4. BASIL. https://basilbiochem.github.io/basil/<br /> <br /> 5. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.<br /> <br /> 6. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.<br /> <br /> 7. 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 Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913<br /> <br /> 8. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.</div>

Michel Evertsen