https://proteopedia.org/api.php?action=feedcontributions&feedformat=atom&user=Jennifer+TaylorProteopedia - User contributions [en]2026-09-15T00:35:11ZUser contributionsMediaWiki 1.43.8https://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_9_MP&diff=2993904User:Jennifer Taylor/Sandbox 9 MP2019-01-23T22:25:08Z<p>Jennifer Taylor: </p>
<hr />
<div>== Structure of GAL4/UAS (Used to control ''Drosophila'' gene expression in Neurology Research) ==<br />
<br />
<applet size='[450,338]' frame='true' align='right'<br />
caption='YYY' /><br />
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<scene name='80/806397/Gal4_bound_to_uas/1'>Gal4 UAS complex</scene><br />
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<scene name='80/806397/P-nitrophenol/1'>p-nitrophenol</scene></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=File:NPO_sdf.pdb&diff=2993903File:NPO sdf.pdb2019-01-23T22:17:04Z<p>Jennifer Taylor: </p>
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<div></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_9_MP&diff=2993902User:Jennifer Taylor/Sandbox 9 MP2019-01-23T22:10:10Z<p>Jennifer Taylor: </p>
<hr />
<div>== Structure of GAL4/UAS (Used to control ''Drosophila'' gene expression in Neurology Research) ==<br />
<br />
<applet size='[450,338]' frame='true' align='right'<br />
caption='YYY' /><br />
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<scene name='80/806397/Gal4_bound_to_uas/1'>Gal4 UAS complex</scene></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_9_MP&diff=2993901User:Jennifer Taylor/Sandbox 9 MP2019-01-23T22:08:52Z<p>Jennifer Taylor: </p>
<hr />
<div>== Structure of GAL4/UAS (Used to control ''Drosophila'' gene expression in Neurology Research) ==<br />
<br />
<<scene name='80/806397/Gal4_bound_to_uas/1'>Gal4 UAS complex</scene>applet size='[450,338]' frame='true' align='right'<br />
caption='YYY' /></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_9_MP&diff=2993900User:Jennifer Taylor/Sandbox 9 MP2019-01-23T21:52:02Z<p>Jennifer Taylor: </p>
<hr />
<div>==Your Heading Here (maybe something like 'Structure')==<br />
<StructureSection load='3COQ' size='340' side='right' caption='Caption for this structure' scene=''><br />
<br />
<applet size='[450,338]' frame='true' align='right'<br />
caption='YYY' /></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_9_MP&diff=2993899User:Jennifer Taylor/Sandbox 9 MP2019-01-23T21:49:19Z<p>Jennifer Taylor: New page: ==Your Heading Here (maybe something like 'Structure')== <StructureSection load='3COQ' size='340' side='right' caption='Caption for this structure' scene=''> This is a default text for you...</p>
<hr />
<div>==Your Heading Here (maybe something like 'Structure')==<br />
<StructureSection load='3COQ' size='340' side='right' caption='Caption for this structure' scene=''><br />
This is a default text for your page '''Jennifer Taylor/Sandbox 9 MP'''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br />
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br />
<br />
== Function ==<br />
<br />
== Disease ==<br />
<br />
== Relevance ==<br />
<br />
== Structural highlights ==<br />
<br />
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> 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 />
</StructureSection><br />
== References ==<br />
<references/></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor&diff=2993898User:Jennifer Taylor2019-01-23T21:46:26Z<p>Jennifer Taylor: </p>
<hr />
<div>*[[User:Jennifer Taylor/Sandbox 1]]<br />
*[[User:Jennifer Taylor/Sandbox 2]]<br />
*[[User:Jennifer Taylor/Sandbox 3]]<br />
*[[User:Jennifer Taylor/Sandbox 4]]<br />
*[[User:Jennifer Taylor/Sandbox 5]]<br />
*[[User:Jennifer Taylor/Sandbox 6]]<br />
*[[User:Jennifer Taylor/Sandbox 7]]<br />
*[[User:Jennifer Taylor/Sandbox 8]]<br />
*[[User:Jennifer Taylor/Sandbox 9_MP]]<br />
*[[User:Jennifer Taylor/Sandbox 10_MP]]<br />
*[[User:Jennifer Taylor/Sandbox 11_MP]]<br />
*[[User:Jennifer Taylor/Sandbox 12_MP]]<br />
*[[User:Jennifer Taylor/Sandbox 13_MP]]<br />
*[[User:Jennifer Taylor/Sandbox 14_MP]]<br />
<br />
<br />
<br />
* Full Real Name: Jennifer L. Taylor<br />
<br />
* Position: Teacher<br />
<br />
* Institution (NO ABBREVIATIONS): Deerfield Academy<br />
<br />
* City, State/Province, Country: Deerfield, MA 01342<br />
<br />
* Field of Expertise or Study: Structural Biology, Ph.D in Chemistry</div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor&diff=2993897User:Jennifer Taylor2019-01-23T21:45:42Z<p>Jennifer Taylor: </p>
<hr />
<div>*[[User:Jennifer Taylor/Sandbox 1]]<br />
*[[User:Jennifer Taylor/Sandbox 2]]<br />
*[[User:Jennifer Taylor/Sandbox 3]]<br />
*[[User:Jennifer Taylor/Sandbox 4]]<br />
*[[User:Jennifer Taylor/Sandbox 5]]<br />
*[[User:Jennifer Taylor/Sandbox 6]]<br />
*[[User:Jennifer Taylor/Sandbox 7]]<br />
*[[User:Jennifer Taylor/Sandbox 8]]<br />
*[[User:Jennifer Taylor/Sandbox 9]]<br />
<br />
<br />
* Full Real Name: Jennifer L. Taylor<br />
<br />
* Position: Teacher<br />
<br />
* Institution (NO ABBREVIATIONS): Deerfield Academy<br />
<br />
* City, State/Province, Country: Deerfield, MA 01342<br />
<br />
* Field of Expertise or Study: Structural Biology, Ph.D in Chemistry</div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor&diff=2993896User:Jennifer Taylor2019-01-23T21:45:28Z<p>Jennifer Taylor: </p>
<hr />
<div>*[[User:Jennifer Taylor/Sandbox 1]]<br />
*[[User:Jennifer Taylor/Sandbox 2]]<br />
*[[User:Jennifer Taylor/Sandbox 3]]<br />
*[[User:Jennifer Taylor/Sandbox 4]]<br />
*[[User:Jennifer Taylor/Sandbox 5]]<br />
*[[User:Jennifer Taylor/Sandbox 6]]<br />
*[[User:Jennifer Taylor/Sandbox 7]]<br />
*[[User:Jennifer Taylor/Sandbox 8]]<br />
*[[User:Jennifer Taylor/Sandbox 9]]<br />
<br />
* Full Real Name: Jennifer L. Taylor<br />
<br />
* Position: Teacher<br />
<br />
* Institution (NO ABBREVIATIONS): Deerfield Academy<br />
<br />
* City, State/Province, Country: Deerfield, MA 01342<br />
<br />
* Field of Expertise or Study: Structural Biology, Ph.D in Chemistry</div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_7&diff=2968448User:Jennifer Taylor/Sandbox 72018-11-07T15:00:22Z<p>Jennifer Taylor: </p>
<hr />
<div>==About 4Q7Q:A Hydrolase==<br />
<StructureSection load='4q7q' size='340' side='both' caption='Caption for this structure' scene=''><br />
This is a default text for your page '''Jennifer Taylor/Sandbox 7'''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br />
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br />
<br />
==What is 4Q7Q?==<br />
In 2000, the protein structure initiative (PSI) began with the goal of finding the three-dimensional structure of as many proteins as possible. In 2010, however, funding for the PSI was terminated. Nevertheless, millions of protein structures were found, and many structures were uncharacterized. One of these uncharacterized proteins was <scene name='78/787195/4q7q_assymetric_assembly/1'>4Q7Q</scene>, which is found in <i>Chitinophaga pinesis</i>. Its molecular weight is 87.1 kDa. Through structural and sequential analysis performed on BLAST, PFam, Dali, and ProMol, 4Q7Q is believed to be a lipase.<br />
==Sequential analysis of 4Q7Q==<br />
<b>BLAST</b>-Sequence homology between 4Q7Q was tested through BLAST; the top hit for 4Q7Q was <scene name='78/787195/4m8k/1'>4M8K</scene>, a GDSL-like lipase. 4Q7Q and 4M8k share a 36% sequence homology, but a low E-value, indicating it is a significant structural match of 4Q7Q. <br />
<br />
<b>PFam</b>-Results from PFam sequence mapping also indicated that 4Q7Q shares sequence homology with other GDSL-like lipases. <br />
<br />
<b>Snapgene</b>-Snapgene was able to reveal the molecular mass of 4Q7Q, later used as a reference when its purified form was run on an SDS-page gel. Snapgene also provided information about tags used to insert its gene into the plasmid used for transformation in DH5α and BL21 E. coli cells.<br />
<br />
[[Image:snapgene_map.png|thumb|left|350px|Figure 1: Snapgene map of 4Q7Q plasmid. 4Q7Q sequence information can be found on the top left corner of the image.]] {{clear}}<br />
== Structural analysis of 4Q7Q==<br />
<b>Dali</b>-Top hits from Dali indicated that 4Q7Q shares structural homology with putative lipases.<br />
<br />
<b>ProMol</b>-Through ProMol, a possible catalytic triad for <scene name='78/787195/4q7q_catalytic_triad/1'>4Q7Q</scene> (Ser164, Asp193, His196) was found due to catalytic triad alignment with 1BRW. 4Q7Q and 1BRW catalytic triad alignment yielded an RMS value of 2.049 and a full structural homology with an RMS value of 4.852.<br />
<br />
==Enzymatic Assay of 4Q7Q==<br />
<b>Reasoning</b>-Results obtained in silico strongly suggested that 4Q7Q was most likely to be hydrolase (and specifically a lipase). Thus, a lipase assay was run on 4Q7Q to confirm its enzymatic function. The lipase assay conducted was done through spectrophotometric methods. Purified 4Q7Q was mixed in solution with P-nitrophenyl butyrate. When nitrophenyl butyrate undergoes hydrolysis, its products are p-nitrophenol and butyrate. P-nitrophenol’s color becomes yellow when this reaction happens in solution. Thus, when a lipase catalyzes the hydrolysis of P-nitrophenol butyrate, a color change in solution will occur. A colorimeter was set at 430nm to detect light in the “yellow” range and thereby measure the change in color occurring when 4Q7Q catalyzed the hydrolysis reaction.<br />
<br />
<b>4.2 Results</b><br />
[[Image:4q7q_SDS.png|thumb|left|350px|Figure 2: SDS-PAGE gel of purified 4Q7Q plasmid. L1: Cell Extract, L2:Flow Through,L3: Wash 3, L4: Elution 1 , L5: Elution 2, L6: Elution 3 , L7: Protein standards.]] {{clear}}<br />
<br />
[[Image:pNPB assay.png|thumb|left|350px|Figure 3:pNPB Assay Results for 4Q7Q.]] {{clear}}<br />
<br />
<b>Conclusion</b>-Due to 4Q7Q’s low concentration found on the SDS-page gel, 4Q7Q to p-Nitrophenol Butyrate (pNPB) ratio in solution was 10:1. A pattern with a logarithmic character was observed during the lipase assay conducted on 4Q7Q, indicating a successful catalysis of a lipid through hydrolysis. <br />
==Future directions==<br />
<b>Cloning</b>-To confirm the suspected catalytic triad of 4Q7Q as suggested by ProMol analysis, site-directed mutagenesis of 4Q7Qs catalytic triad was performed through PCR. Substitutions of residues were completed as followed: Ser164Ala, Asp193Ala, His196Ala. Following a successful cloning, a p-NPB lipase assay will be performed to see whether the new mutations on 4Q7Q deterred catalysis of the hydrolysis reaction of p-NPB. A deterred would indicate the cloned sequences did, in fact, mutate the catalytic triad, altering the structure of the cloned 4Q7Q and affecting enzymatic activity. <br />
<br />
[[Image:4Q7Q_Mutagenesis.png|thumb|left|350px|Figure 3:Mutagenesis of 4Q7Q (Ser164Ala, Asp193Ala, His196Ala).]] {{clear}}<br />
<br />
<b>Enzyme Kinetics of 4Q7Q</b>=Further data of enzyme activity of 4Q7Q using our lipase assay at different concentrations of p-NPB can be performed to create a Lineweaver-Burk plot that would deepen analysis and understanding of the enzymatic activity of 4Q7Q.<br />
<br />
<b>Optimization of enzymatic activity</b>-Conditions for optimized enzyme activity can be tested by performing iterations of the p-NPB assay by altering environmental conditions of the enzyme (pH and temperature).<br />
<br />
<br />
<br />
</StructureSection><br />
== References and Notes ==<br />
<references/></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_7&diff=2960165User:Jennifer Taylor/Sandbox 72018-10-17T21:34:15Z<p>Jennifer Taylor: </p>
<hr />
<div>==About 4Q7Q:A Hydrolase==<br />
<StructureSection load='4q7q' size='340' side='both' caption='Caption for this structure' scene=''><br />
This is a default text for your page '''Jennifer Taylor/Sandbox 7'''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br />
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br />
<br />
==What is 4Q7Q?==<br />
In 2000, the protein structure initiative (PSI) began with the goal of finding the three-dimensional structure of as many proteins as possible. In 2010, however, funding for the PSI was terminated. Nevertheless, millions of protein structures were found, and many structures were uncharacterized. One of these uncharacterized proteins was <scene name='78/787195/4q7q_assymetric_assembly/1'>4Q7Q</scene>, which is found in <i>Chitinophaga pinesis</i>. Its molecular weight is 87.1 kDa. Through structural and sequential analysis performed on BLAST, PFam, Dali, and ProMol, 4Q7Q is believed to be a lipase.<br />
==Sequential analysis of 4Q7Q==<br />
<b>BLAST</b>-Sequence homology between 4Q7Q was tested through BLAST; the top hit for 4Q7Q was <scene name='78/787195/4m8k/1'>4M8K</scene>, a GDSL-like lipase. 4Q7Q and 4M8k share a 36% sequence homology, but a low E-value, indicating it is a significant structural match of 4Q7Q. <br />
<br />
<b>PFam</b>-Results from PFam sequence mapping also indicated that 4Q7Q shares sequence homology with other GDSL-like lipases. <br />
<br />
<b>Snapgene</b>-Snapgene was able to reveal the molecular mass of 4Q7Q, later used as a reference when its purified form was run on an SDS-page gel. Snapgene also provided information about tags used to insert its gene into the plasmid used for transformation in DH5α and BL21 E. coli cells.<br />
<br />
[[Image:snapgene_map.png|thumb|left|350px|Figure 1: Snapgene map of 4Q7Q plasmid. 4Q7Q sequence information can be found on the top left corner of the image.]] {{clear}}<br />
== Structural analysis of 4Q7Q==<br />
<b>Dali</b>-Top hits from Dali indicated that 4Q7Q shares structural homology with putative lipases.<br />
<br />
<b>ProMol</b>-Through ProMol, a possible catalytic triad for <scene name='78/787195/4q7q_catalytic_triad/1'>4Q7Q</scene> (Ser164, Asp193, His196) was found due to catalytic triad alignment with 1BRW. 4Q7Q and 1BRW catalytic triad alignment yielded an RMS value of 2.049 and a full structural homology with an RMS value of 4.852.<br />
<br />
==Enzymatic Assay of 4Q7Q==<br />
<b>Reasoning</b>-Results obtained in silico strongly suggested that 4Q7Q was most likely to be hydrolase (and specifically a lipase). Thus, a lipase assay was run on 4Q7Q to confirm its enzymatic function. The lipase assay conducted was done through spectrophotometric methods. Purified 4Q7Q was mixed in solution with P-nitrophenyl butyrate. When nitrophenyl butyrate undergoes hydrolysis, its products are p-nitrophenol and butyrate. P-nitrophenol’s color becomes yellow when this reaction happens in solution. Thus, when a lipase catalyzes the hydrolysis of P-nitrophenol butyrate, a color change in solution will occur. A colorimeter was set at 430nm to detect light in the “yellow” range and thereby measure the change in color occurring when 4Q7Q catalyzed the hydrolysis reaction.<br />
<br />
<b>4.2 Results</b><br />
[[Image:4q7q_SDS.png|thumb|left|350px|Figure 2: SDS-PAGE gel of purified 4Q7Q plasmid. L1: Elution 1, L2: Elution2, L4: , L5: , L6: , L7: L8: Protein standards.]] {{clear}}<br />
<br />
[[Image:pNPB assay.png|thumb|left|350px|Figure 3:pNPB Assay Results for 4Q7Q.]] {{clear}}<br />
<br />
<b>Conclusion</b>-Due to 4Q7Q’s low concentration found on the SDS-page gel, 4Q7Q to p-Nitrophenol Butyrate (pNPB) ratio in solution was 10:1. A pattern with a logarithmic character was observed during the lipase assay conducted on 4Q7Q, indicating a successful catalysis of a lipid through hydrolysis. <br />
==Future directions==<br />
<b>Cloning</b>-To confirm the suspected catalytic triad of 4Q7Q as suggested by ProMol analysis, site-directed mutagenesis of 4Q7Qs catalytic triad was performed through PCR. Substitutions of residues were completed as followed: Ser164Ala, Asp193Ala, His196Ala. Following a successful cloning, a p-NPB lipase assay will be performed to see whether the new mutations on 4Q7Q deterred catalysis of the hydrolysis reaction of p-NPB. A deterred would indicate the cloned sequences did, in fact, mutate the catalytic triad, altering the structure of the cloned 4Q7Q and affecting enzymatic activity. <br />
<br />
[[Image:4Q7Q_Mutagenesis.png|thumb|left|350px|Figure 3:Mutagenesis of 4Q7Q (Ser164Ala, Asp193Ala, His196Ala).]] {{clear}}<br />
<br />
<b>Enzyme Kinetics of 4Q7Q</b>=Further data of enzyme activity of 4Q7Q using our lipase assay at different concentrations of p-NPB can be performed to create a Lineweaver-Burk plot that would deepen analysis and understanding of the enzymatic activity of 4Q7Q.<br />
<br />
<b>Optimization of enzymatic activity</b>-Conditions for optimized enzyme activity can be tested by performing iterations of the p-NPB assay by altering environmental conditions of the enzyme (pH and temperature).<br />
<br />
<br />
<br />
</StructureSection><br />
== References and Notes ==<br />
<references/></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_7&diff=2960164User:Jennifer Taylor/Sandbox 72018-10-17T21:18:01Z<p>Jennifer Taylor: </p>
<hr />
<div>==About 4Q7Q:A Hydrolase==<br />
<StructureSection load='4q7q' size='340' side='both' caption='Caption for this structure' scene=''><br />
This is a default text for your page '''Jennifer Taylor/Sandbox 7'''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br />
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br />
<br />
==What is 4Q7Q?==<br />
In 2000, the protein structure initiative (PSI) began with the goal of finding the three-dimensional structure of as many proteins as possible. In 2010, however, funding for the PSI was terminated. Nevertheless, millions of protein structures were found, and many structures were uncharacterized. One of these uncharacterized proteins was <scene name='78/787195/4q7q_assymetric_assembly/1'>4Q7Q</scene>, which is found in <i>Chitinophaga pinesis</i>. Its molecular weight is 87.1 kDa. Through structural and sequential analysis performed on BLAST, PFam, Dali, and ProMol, 4Q7Q is believed to be a lipase.<br />
==Sequential analysis of 4Q7Q==<br />
<b>BLAST</b>-Sequence homology between 4Q7Q was tested through BLAST; the top hit for 4Q7Q was <scene name='78/787195/4m8k/1'>4M8K</scene>, a GDSL-like lipase. 4Q7Q and 4M8k share a 36% sequence homology, but a low E-value, indicating it is a significant structural match of 4Q7Q. <br />
<br />
<b>PFam</b>-Results from PFam sequence mapping also indicated that 4Q7Q shares sequence homology with other GDSL-like lipases. <br />
<br />
<b>Snapgene</b>-Snapgene was able to reveal the molecular mass of 4Q7Q, later used as a reference when its purified form was run on an SDS-page gel. Snapgene also provided information about tags used to insert its gene into the plasmid used for transformation in DH5α and BL21 E. coli cells.<br />
<br />
[[Image:snapgene_map.png|thumb|left|350px|Figure 1: Snapgene map of 4Q7Q plasmid. 4Q7Q sequence information can be found on the top left corner of the image.]] {{clear}}<br />
== Structural analysis of 4Q7Q==<br />
<b>Dali</b>-Top hits from Dali indicated that 4Q7Q shares structural homology with putative lipases.<br />
<br />
<b>ProMol</b>-Through ProMol, a possible catalytic triad for <scene name='78/787195/4q7q_catalytic_triad/1'>4Q7Q</scene> (Ser164, Asp193, His196) was found due to catalytic triad alignment with 1BRW. 4Q7Q and 1BRW catalytic triad alignment yielded an RMS value of 2.049 and a full structural homology with an RMS value of 4.852.<br />
<br />
==Enzymatic Assay of 4Q7Q==<br />
<b>Reasoning</b>-Results obtained in silico strongly suggested that 4Q7Q was most likely to be hydrolase (and specifically a lipase). Thus, a lipase assay was run on 4Q7Q to confirm its enzymatic function. The lipase assay conducted was done through spectrophotometric methods. Purified 4Q7Q was mixed in solution with P-nitrophenyl butyrate. When nitrophenyl butyrate undergoes hydrolysis, its products are p-nitrophenol and butyrate. P-nitrophenol’s color becomes yellow when this reaction happens in solution. Thus, when a lipase catalyzes the hydrolysis of P-nitrophenol butyrate, a color change in solution will occur. A colorimeter was set at 430nm to detect light in the “yellow” range and thereby measure the change in color occurring when 4Q7Q catalyzed the hydrolysis reaction.<br />
<br />
<b>4.2 Results</b><br />
[[Image:4q7q_SDS.png|thumb|left|350px|Figure 2: SDS-PAGE gel of purified 4Q7Q plasmid.]] {{clear}}<br />
<br />
[[Image:pNPB assay.png|thumb|left|350px|Figure 3:pNPB Assay Results for 4Q7Q.]] {{clear}}<br />
<br />
<b>Conclusion</b>-Due to 4Q7Q’s low concentration found on the SDS-page gel, 4Q7Q to p-Nitrophenol Butyrate (pNPB) ratio in solution was 10:1. A pattern with a logarithmic character was observed during the lipase assay conducted on 4Q7Q, indicating a successful catalysis of a lipid through hydrolysis. <br />
==Future directions==<br />
<b>Cloning</b>-To confirm the suspected catalytic triad of 4Q7Q as suggested by ProMol analysis, site-directed mutagenesis of 4Q7Qs catalytic triad was performed through PCR. Substitutions of residues were completed as followed: Ser164Ala, Asp193Ala, His196Ala. Following a successful cloning, a p-NPB lipase assay will be performed to see whether the new mutations on 4Q7Q deterred catalysis of the hydrolysis reaction of p-NPB. A deterred would indicate the cloned sequences did, in fact, mutate the catalytic triad, altering the structure of the cloned 4Q7Q and affecting enzymatic activity. <br />
<br />
[[Image:4Q7Q_Mutagenesis.png|thumb|left|350px|Figure 3:Mutagenesis of 4Q7Q (Ser164Ala, Asp193Ala, His196Ala).]] {{clear}}<br />
<br />
<b>Enzyme Kinetics of 4Q7Q</b>=Further data of enzyme activity of 4Q7Q using our lipase assay at different concentrations of p-NPB can be performed to create a Lineweaver-Burk plot that would deepen analysis and understanding of the enzymatic activity of 4Q7Q.<br />
<br />
<b>Optimization of enzymatic activity</b>-Conditions for optimized enzyme activity can be tested by performing iterations of the p-NPB assay by altering environmental conditions of the enzyme (pH and temperature).<br />
<br />
<br />
<br />
</StructureSection><br />
== References and Notes ==<br />
<references/></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_6&diff=2904807User:Jennifer Taylor/Sandbox 62018-05-26T21:17:01Z<p>Jennifer Taylor: </p>
<hr />
<div>== Overview ==<br />
[[Image: Screen_Shot_2018-05-21_at_10.20.55.png|thumb||left|900pxl|Figure 1:Diagrams Figure 1 (A-D clockwise) A: Hydrolases simply refer to any enzyme that catalyzes a hydrolysis reaction where H20 is added to cleave chemical bonds. Figure B: Esterases split esters into an alcohol and an acid through hydrolysis Figure C: Lipases catalyze the hydrolysis of lipids, or fats. Lipases are most commonly found on ∂/ß hydrolase chain and often contain the catalytic triad serine, histidine, and aspartic acid. Figure D: Lipid Chain with an extend- ed hydrocarbon chain, similar to the nitrophenyl palmitate.]] Throughout the course of this past year, we attempted to determine the function of our protein 2QRU. Protein shape and protein function are really closely related, so if two proteins that look similar are compared, there is a high likelihood that they do the same thing. This was the underlying basis for our project. After initial computer analysis, we found that many of the proteins that had structural similarity to 2QRU were esterases. Thus, our preliminary hypothesis predicted that 2QRU would be a lipase, a subclass of esterases. We performed an esterase assay to prove that 2QRU was an esterase first, and are yet to determine if it can be classified as a lipase.<br />
<br />
== Background ==<br />
Proteins are one of four major macromolecules in biology. Present in nearly every living organism, proteins have a diverse set of functions ranging from regulating cell activity to catalyze reactions. Due to the sheer number of proteins in existence, there still remain many to discover and characterize. In 2000, the Protein Structure Initiative began an attempt to solve 3D-structures of proteins with known sequences in order to understand their functions. Though the initiative was successful, they faced financial drawbacks in 2015. There still remain several protein structures with unknown functions in a public database called the Protein Data Bank. In a final PSI summary published in 2017, 6920 structures had been solved in their seventeen years of work. What we tried to do is take one of the protein structures solved by the PSI and characterize its function.<br />
<StructureSection load='2qru' size='340' side='right' caption='Caption for this structure' scene=''><br />
== Initial Process ==<br />
The first thing we did was try to see if we could express our protein in a cell. We first tried to insert our plasmid containing 2QRU into DH5∂ cells but found that this cell didn't produce the T7 polymerase that was necessary to transcribe 2QRU. We then switched to E. Coli cells. We inserted our plasmid into E. Coli using a standard transformation protocol. We then measured the concentration of our plasmid and then ran an SDS page gel to determine If our plasmid was successfully expressed in our cells. After that, we purified our protein or essentially squeezed it out of our cells to get protein concentrate. We did this using His-Pur Nickel-NTA Spin columns. We put our cell extract into a column, added a specific buffer, and then centrifuged the column. All of the non-protein substances in the cell were filtered out to the bottom of the column in the first few washes. This meant that by our fifth or six wash, the substances coming through the column were our protein, 2QRU. We then ran a gel with our protein extract to see if it was successfully expressed. What we found, as expected, was that the band at our protein weight got stronger as the purification process went on, meaning the sample from our first wash had many bands signifying other proteins in the cell, not just ours. But, the sample from our last wash had 1 clean band at the expected weight of our protein, 33.8 kda. [[Image:Screen Shot 2018-05-21 at 10.18.50.png|thumb||left|500pxl|Figure 1:This is our protein gel]] Figure 1 is an SDS Page Gel showing the molecular weight of the puri ed 2QRU from February. The ladder is in kDa. [Lane 8 – Ladder (5 uL), Lane 9 – Cell ex- tract (15 uL), Lane 10 – Flow through 6 (15 uL), Lane 11 – Wash 1 (15 uL), Lane 12 – Wash 3 (15 uL), Lane 13 – Elution 1 (14 uL), Lane 14 – Elution 2 (14 uL), Lane 15 – Elution 3 (14 uL)]<br />
</StructureSection><br />
== Structural Analysis ==<br />
[[Image:EfCD00584424 Map.png|thumb||left|500pxl|Figure 2:This is our plasmid visualized using Snap Gene]] Figure 2 shows what my protein looks like inside a plasmid. We used SnapGene to figure out the weight of our protein 33.8 kda. Once we proved that we could produce our protein, we started to think about how we wanted to characterize its functions. Since we know that function and structure go hand in hand, we also used a program called PyMol to first look at the 3D structure of our protein. Here is a visualization of my protein highlighting the <scene name='78/787197/1_2qru_cartoon/1'>alpha helices and beta sheets.</scene> It’s an alpha/beta hydrolase that has one chain. It's 816 base pairs long. Then, using Dali and pFam, we ran a search for other proteins that were structurally similar to ours. We found a few hits including 1TAH and 1C4X that are classified as esterases by aligning the active sites of these proteins to ours. [[Image:Screen Shot 2018-05-21 at 10.20.32.png|thumb||left|750pxl|Figure 3:These are our PyMol alignments]] The left most photo in Figure 3 shows the alignment of the active sites of 2QRU and 1TAH in PyMol. The RMS values of 1TAH’s catalytic triads compared to 2QRU’s catalytic triad was much lower than the RMS value of the full length alignment so this alignment is a better representation of the structural similarity. The middle photo showss the alignment between 2QRU and1C4X in PyMol. The RMS values of 1C4X’s catalytic triads compared to 2QRU’s catalytic triad was much lower than the RMS value of the full length alignment so this alignment is a better representation of the struc- tural similarity. The right most photo shows the alignment of 2QRU and 3FAK structures in PyMol.The RMS of 3FAK and 2QRU’s active site was much higher than the full structure alignment RMS, so this alignment is a better representation of the structural similarity.<br />
<br />
== Assays ==<br />
In order to determine if our protein was an esterase, we used an assay found in a published paper that sought to characterize 3FAK as an esterase. They performed a colorimetric assay which we modified for our study. The study we researched used p-nitrophenyl butyrate to test if 3FAK was an esterase. When the protein came into contact with this liquid, the entire solution turned yellow. We made a blank cuvette with Tris buffer and p-nitrophenyl butyrate. Then we added our protein and measured how the color of the solution changed over 30 sec intervals for 2 min. We repeated this using various concentrations of protein. <br />
<br />
== Results ==<br />
[[Image:Screen Shot 2018-05-21 at 10.20.12.png|thumb||left|1000pxl|Figure 4:This is a graph summerizing our assay results.]] Figure 4 shows the results of the successful Esterase Assay with varied concentrations of NPB dissolved in n-Heptane. The change in colorimeter absorbance over time is shown. We graphed the OD readings for each concentration and found that 0.375M of 2QRU produced the highest rate of reaction with p-nitrophenyl butyrate. We can first conclude that our protein is an esterase, but our results were slightly confusing. We expected that the highest concentration of PNB tested, 0.5M, would have the fastest reaction rate. However, we since the second highest concentration produced the highest reaction rate, we thought perhaps 0.375M could be the optimal concentration. <br />
Possible errors include inconsistent timing when inserting the induced cuvette into the colorimeter. This may explain why 0.375M of p-nitrophenyl butyrate had the steepest reaction rate with 2QRU even though we ran tests with higher concentrations of p-nitrophenyl butyrate. P-nitrophenyl butyrate concentrations below 0.15M failed to produce a measurable reaction with 2QRU. Thus, we tried the assay again with higher concentrations of p-nitrophenyl butyrate. Another error occurred at the beginning of our research to prove that 2QRU is an esterase. Another error occurred we performed an enzymatic assay of an esterase from Sigma Aldrich that tracked the enzymatic reaction by measuring the pH change over time. After attempting the assay, there were no results and therefore we could not characterize 2QRU. This lead us to our successful assay.<br />
<br />
== Future Directions ==<br />
After coming to the conclusion that 2QRU is an esterase, our next research activity would be to test if 2QRU is also a lipase. Three students from The Pingry School in New Jersey per- formed a lipase assay with 2QRU using nitrophenyl palmitate rather than p-nitrophenyl butyrate to determine if 2QRU is a lipase. Nitrophenyl palmitate has (CH2)14 side chain than nitrophenyl butyrate, so the protein has to act on a bigger substrate. If this assay works for us, then would be able to conclude that 2QRU is also a lipase. Another avenue to explore after con rming 2QRU is a lipase is mutagenesis. If we mutated the catalytic triad responsible for the reaction with nitrophenyl palmitate and ran the lipase assay again, we should see no reaction occur in the cuvette. We can use this to con rm that 2QRU is a lipase. The same thing can be done to confirm 2QRU is an esterase if we run the esterase assay using p-nitrophenyl butyrate.<br />
<br />
== References ==<br />
[1]Alberts, Bruce. Molecular Biology of the Cell. 6th ed. New York, NY: Garland Science, Taylor and Francis Group, 2015.<br />
[2] Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) “Basic local alignment search tool.” J. Mol. Biol. 215:403-410. PubMed.<br />
[3] “Enzymatic Assay of an Esterase.” Sigma-Aldrich. Accessed April 27, 2018. https://www.sigmaaldrich.com/technical-documents/protocols/biology/enzy- matic-assay-of-esterase.html.<br />
[4] Lisa Holm; Laura M. Laakso (2016) Dali server update. Nucleic acids research 44 (W1), W351-W355. PDF<br />
[5]“Milestones Tables.” PSI Structural Biology Knowledgebase. Accessed April 27, 2018. http://targetdb.pdb.org/Metrics/MilestonesTables.html.<br />
[6] Nam, Ki Hyun, Min-Young Kim, Soo-Jin Kim, Amit Pryadarshi, Won Ho Lee, and KwangYeon Hwang. “Structural and Functional Analysis of a Novel EstE5 Belonging to the Subfamily of Hormone-Sensitive Lipase.” Elsevier, December 29, 2008. https://www.ncbi.nlm.nih.gov/pubmed/19116143.<br />
[7] PDB: 2QRU Cuff, M.E., Volkart, L., Moy, S., Joachimiak, A.Structure of an alpha/beta hydrolase superfamily protein from Enterococcus faecalis.<br />
[8] ProMol Project. Dr. Paul Craig, Dr. Herbert Bernstein, Dr. Jeff Mills at Rochester Institute of Technology.<br />
[9] SnapGene Software (from GSL Biotech; available at snapgene.com).<br />
[10] The Pfam protein families database: towards a more sustainable future: R.D. Finn, P. Coggill, R.Y. Eberhardt, S.R. Eddy, J. Mistry, A.L. Mitchell, S.C. Potter, M. Punta, M. Qureshi, A. Sangrador-Vegas, G.A. Salazar, J. Tate, A. BatemanNucleic Acids Research (2016) Database Issue 44:D279-D285<br />
[11] The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.<br />
[12] Zhong, Qixin and Charles E. Glatz. “Enzymatic Assay Method for Evaluating the Lipase<br />
Activity in Complex Extracts from Transgenic Corn Seed.” Journal of Agricultural and Food Chemistry. 2006(54): 3181-3185. https://lib.dr.iastate.edu/cgi/view- content.cgi?referer=&httpsredir=1&article=1075&context=cbe_pubs.<br />
[13] Messaoudi, Abdelmonem & Belguith, Hatem & Gram, Imen & Ben Hamida, Jeannette. (2010). Classi cation of EC 3.1.1.3 bacterial true lipases using phyloge- netic analysis. African Journal of Biotechnology. 9. 8243-8247. 10.5897/AJB10.721.</div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_6&diff=2904806User:Jennifer Taylor/Sandbox 62018-05-26T21:15:32Z<p>Jennifer Taylor: </p>
<hr />
<div>== Overview ==<br />
Throughout the course of this past year, we attempted to determine the function of our protein 2QRU. Protein shape and protein function are really closely related, so if two proteins that look similar are compared, there is a high likelihood that they do the same thing. This was the underlying basis for our project. After initial computer analysis, we found that many of the proteins that had structural similarity to 2QRU were esterases. Thus, our preliminary hypothesis predicted that 2QRU would be a lipase, a subclass of esterases. We performed an esterase assay to prove that 2QRU was an esterase first, and are yet to determine if it can be classified as a lipase. [[Image: Screen_Shot_2018-05-21_at_10.20.55.png||left|Figure 1:Diagrams Figure 1 (A-D clockwise) A: Hydrolases simply refer to any enzyme that catalyzes a hydrolysis reaction where H20 is added to cleave chemical bonds. Figure B: Esterases split esters into an alcohol and an acid through hydrolysis Figure C: Lipases catalyze the hydrolysis of lipids, or fats. Lipases are most commonly found on ∂/ß hydrolase chain and often contain the catalytic triad serine, histidine, and aspartic acid. Figure D: Lipid Chain with an extend- ed hydrocarbon chain, similar to the nitrophenyl palmitate.]]<br />
<br />
== Background ==<br />
Proteins are one of four major macromolecules in biology. Present in nearly every living organism, proteins have a diverse set of functions ranging from regulating cell activity to catalyze reactions. Due to the sheer number of proteins in existence, there still remain many to discover and characterize. In 2000, the Protein Structure Initiative began an attempt to solve 3D-structures of proteins with known sequences in order to understand their functions. Though the initiative was successful, they faced financial drawbacks in 2015. There still remain several protein structures with unknown functions in a public database called the Protein Data Bank. In a final PSI summary published in 2017, 6920 structures had been solved in their seventeen years of work. What we tried to do is take one of the protein structures solved by the PSI and characterize its function.<br />
<StructureSection load='2qru' size='340' side='right' caption='Caption for this structure' scene=''><br />
== Initial Process ==<br />
The first thing we did was try to see if we could express our protein in a cell. We first tried to insert our plasmid containing 2QRU into DH5∂ cells but found that this cell didn't produce the T7 polymerase that was necessary to transcribe 2QRU. We then switched to E. Coli cells. We inserted our plasmid into E. Coli using a standard transformation protocol. We then measured the concentration of our plasmid and then ran an SDS page gel to determine If our plasmid was successfully expressed in our cells. After that, we purified our protein or essentially squeezed it out of our cells to get protein concentrate. We did this using His-Pur Nickel-NTA Spin columns. We put our cell extract into a column, added a specific buffer, and then centrifuged the column. All of the non-protein substances in the cell were filtered out to the bottom of the column in the first few washes. This meant that by our fifth or six wash, the substances coming through the column were our protein, 2QRU. We then ran a gel with our protein extract to see if it was successfully expressed. What we found, as expected, was that the band at our protein weight got stronger as the purification process went on, meaning the sample from our first wash had many bands signifying other proteins in the cell, not just ours. But, the sample from our last wash had 1 clean band at the expected weight of our protein, 33.8 kda. [[Image:Screen Shot 2018-05-21 at 10.18.50.png|thumb||left|500pxl|Figure 1:This is our protein gel]] Figure 1 is an SDS Page Gel showing the molecular weight of the puri ed 2QRU from February. The ladder is in kDa. [Lane 8 – Ladder (5 uL), Lane 9 – Cell ex- tract (15 uL), Lane 10 – Flow through 6 (15 uL), Lane 11 – Wash 1 (15 uL), Lane 12 – Wash 3 (15 uL), Lane 13 – Elution 1 (14 uL), Lane 14 – Elution 2 (14 uL), Lane 15 – Elution 3 (14 uL)]<br />
</StructureSection><br />
== Structural Analysis ==<br />
[[Image:EfCD00584424 Map.png|thumb||left|500pxl|Figure 2:This is our plasmid visualized using Snap Gene]] Figure 2 shows what my protein looks like inside a plasmid. We used SnapGene to figure out the weight of our protein 33.8 kda. Once we proved that we could produce our protein, we started to think about how we wanted to characterize its functions. Since we know that function and structure go hand in hand, we also used a program called PyMol to first look at the 3D structure of our protein. Here is a visualization of my protein highlighting the <scene name='78/787197/1_2qru_cartoon/1'>alpha helices and beta sheets.</scene> It’s an alpha/beta hydrolase that has one chain. It's 816 base pairs long. Then, using Dali and pFam, we ran a search for other proteins that were structurally similar to ours. We found a few hits including 1TAH and 1C4X that are classified as esterases by aligning the active sites of these proteins to ours. [[Image:Screen Shot 2018-05-21 at 10.20.32.png|thumb||left|750pxl|Figure 3:These are our PyMol alignments]] The left most photo in Figure 3 shows the alignment of the active sites of 2QRU and 1TAH in PyMol. The RMS values of 1TAH’s catalytic triads compared to 2QRU’s catalytic triad was much lower than the RMS value of the full length alignment so this alignment is a better representation of the structural similarity. The middle photo showss the alignment between 2QRU and1C4X in PyMol. The RMS values of 1C4X’s catalytic triads compared to 2QRU’s catalytic triad was much lower than the RMS value of the full length alignment so this alignment is a better representation of the struc- tural similarity. The right most photo shows the alignment of 2QRU and 3FAK structures in PyMol.The RMS of 3FAK and 2QRU’s active site was much higher than the full structure alignment RMS, so this alignment is a better representation of the structural similarity.<br />
<br />
== Assays ==<br />
In order to determine if our protein was an esterase, we used an assay found in a published paper that sought to characterize 3FAK as an esterase. They performed a colorimetric assay which we modified for our study. The study we researched used p-nitrophenyl butyrate to test if 3FAK was an esterase. When the protein came into contact with this liquid, the entire solution turned yellow. We made a blank cuvette with Tris buffer and p-nitrophenyl butyrate. Then we added our protein and measured how the color of the solution changed over 30 sec intervals for 2 min. We repeated this using various concentrations of protein. <br />
<br />
== Results ==<br />
[[Image:Screen Shot 2018-05-21 at 10.20.12.png|thumb||left|1000pxl|Figure 4:This is a graph summerizing our assay results.]] Figure 4 shows the results of the successful Esterase Assay with varied concentrations of NPB dissolved in n-Heptane. The change in colorimeter absorbance over time is shown. We graphed the OD readings for each concentration and found that 0.375M of 2QRU produced the highest rate of reaction with p-nitrophenyl butyrate. We can first conclude that our protein is an esterase, but our results were slightly confusing. We expected that the highest concentration of PNB tested, 0.5M, would have the fastest reaction rate. However, we since the second highest concentration produced the highest reaction rate, we thought perhaps 0.375M could be the optimal concentration. <br />
Possible errors include inconsistent timing when inserting the induced cuvette into the colorimeter. This may explain why 0.375M of p-nitrophenyl butyrate had the steepest reaction rate with 2QRU even though we ran tests with higher concentrations of p-nitrophenyl butyrate. P-nitrophenyl butyrate concentrations below 0.15M failed to produce a measurable reaction with 2QRU. Thus, we tried the assay again with higher concentrations of p-nitrophenyl butyrate. Another error occurred at the beginning of our research to prove that 2QRU is an esterase. Another error occurred we performed an enzymatic assay of an esterase from Sigma Aldrich that tracked the enzymatic reaction by measuring the pH change over time. After attempting the assay, there were no results and therefore we could not characterize 2QRU. This lead us to our successful assay.<br />
<br />
== Future Directions ==<br />
After coming to the conclusion that 2QRU is an esterase, our next research activity would be to test if 2QRU is also a lipase. Three students from The Pingry School in New Jersey per- formed a lipase assay with 2QRU using nitrophenyl palmitate rather than p-nitrophenyl butyrate to determine if 2QRU is a lipase. Nitrophenyl palmitate has (CH2)14 side chain than nitrophenyl butyrate, so the protein has to act on a bigger substrate. If this assay works for us, then would be able to conclude that 2QRU is also a lipase. Another avenue to explore after con rming 2QRU is a lipase is mutagenesis. If we mutated the catalytic triad responsible for the reaction with nitrophenyl palmitate and ran the lipase assay again, we should see no reaction occur in the cuvette. We can use this to con rm that 2QRU is a lipase. The same thing can be done to confirm 2QRU is an esterase if we run the esterase assay using p-nitrophenyl butyrate.<br />
<br />
== References ==<br />
[1]Alberts, Bruce. Molecular Biology of the Cell. 6th ed. New York, NY: Garland Science, Taylor and Francis Group, 2015.<br />
[2] Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) “Basic local alignment search tool.” J. Mol. Biol. 215:403-410. PubMed.<br />
[3] “Enzymatic Assay of an Esterase.” Sigma-Aldrich. Accessed April 27, 2018. https://www.sigmaaldrich.com/technical-documents/protocols/biology/enzy- matic-assay-of-esterase.html.<br />
[4] Lisa Holm; Laura M. Laakso (2016) Dali server update. Nucleic acids research 44 (W1), W351-W355. PDF<br />
[5]“Milestones Tables.” PSI Structural Biology Knowledgebase. Accessed April 27, 2018. http://targetdb.pdb.org/Metrics/MilestonesTables.html.<br />
[6] Nam, Ki Hyun, Min-Young Kim, Soo-Jin Kim, Amit Pryadarshi, Won Ho Lee, and KwangYeon Hwang. “Structural and Functional Analysis of a Novel EstE5 Belonging to the Subfamily of Hormone-Sensitive Lipase.” Elsevier, December 29, 2008. https://www.ncbi.nlm.nih.gov/pubmed/19116143.<br />
[7] PDB: 2QRU Cuff, M.E., Volkart, L., Moy, S., Joachimiak, A.Structure of an alpha/beta hydrolase superfamily protein from Enterococcus faecalis.<br />
[8] ProMol Project. Dr. Paul Craig, Dr. Herbert Bernstein, Dr. Jeff Mills at Rochester Institute of Technology.<br />
[9] SnapGene Software (from GSL Biotech; available at snapgene.com).<br />
[10] The Pfam protein families database: towards a more sustainable future: R.D. Finn, P. Coggill, R.Y. Eberhardt, S.R. Eddy, J. Mistry, A.L. Mitchell, S.C. Potter, M. Punta, M. Qureshi, A. Sangrador-Vegas, G.A. Salazar, J. Tate, A. BatemanNucleic Acids Research (2016) Database Issue 44:D279-D285<br />
[11] The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.<br />
[12] Zhong, Qixin and Charles E. Glatz. “Enzymatic Assay Method for Evaluating the Lipase<br />
Activity in Complex Extracts from Transgenic Corn Seed.” Journal of Agricultural and Food Chemistry. 2006(54): 3181-3185. https://lib.dr.iastate.edu/cgi/view- content.cgi?referer=&httpsredir=1&article=1075&context=cbe_pubs.<br />
[13] Messaoudi, Abdelmonem & Belguith, Hatem & Gram, Imen & Ben Hamida, Jeannette. (2010). Classi cation of EC 3.1.1.3 bacterial true lipases using phyloge- netic analysis. African Journal of Biotechnology. 9. 8243-8247. 10.5897/AJB10.721.</div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_6&diff=2904805User:Jennifer Taylor/Sandbox 62018-05-26T21:14:23Z<p>Jennifer Taylor: </p>
<hr />
<div>== Overview ==<br />
Throughout the course of this past year, we attempted to determine the function of our protein 2QRU. Protein shape and protein function are really closely related, so if two proteins that look similar are compared, there is a high likelihood that they do the same thing. This was the underlying basis for our project. After initial computer analysis, we found that many of the proteins that had structural similarity to 2QRU were esterases. Thus, our preliminary hypothesis predicted that 2QRU would be a lipase, a subclass of esterases. We performed an esterase assay to prove that 2QRU was an esterase first, and are yet to determine if it can be classified as a lipase. [[Image: Screen_Shot_2018-05-21_at_10.20.55.png|Figure 1:Diagrams Figure 1 (A-D clockwise) A: Hydrolases simply refer to any enzyme that catalyzes a hydrolysis reaction where H20 is added to cleave chemical bonds. Figure B: Esterases split esters into an alcohol and an acid through hydrolysis Figure C: Lipases catalyze the hydrolysis of lipids, or fats. Lipases are most commonly found on ∂/ß hydrolase chain and often contain the catalytic triad serine, histidine, and aspartic acid. Figure D: Lipid Chain with an extend- ed hydrocarbon chain, similar to the nitrophenyl palmitate.]]<br />
<br />
== Background ==<br />
Proteins are one of four major macromolecules in biology. Present in nearly every living organism, proteins have a diverse set of functions ranging from regulating cell activity to catalyze reactions. Due to the sheer number of proteins in existence, there still remain many to discover and characterize. In 2000, the Protein Structure Initiative began an attempt to solve 3D-structures of proteins with known sequences in order to understand their functions. Though the initiative was successful, they faced financial drawbacks in 2015. There still remain several protein structures with unknown functions in a public database called the Protein Data Bank. In a final PSI summary published in 2017, 6920 structures had been solved in their seventeen years of work. What we tried to do is take one of the protein structures solved by the PSI and characterize its function.<br />
<StructureSection load='2qru' size='340' side='right' caption='Caption for this structure' scene=''><br />
== Initial Process ==<br />
The first thing we did was try to see if we could express our protein in a cell. We first tried to insert our plasmid containing 2QRU into DH5∂ cells but found that this cell didn't produce the T7 polymerase that was necessary to transcribe 2QRU. We then switched to E. Coli cells. We inserted our plasmid into E. Coli using a standard transformation protocol. We then measured the concentration of our plasmid and then ran an SDS page gel to determine If our plasmid was successfully expressed in our cells. After that, we purified our protein or essentially squeezed it out of our cells to get protein concentrate. We did this using His-Pur Nickel-NTA Spin columns. We put our cell extract into a column, added a specific buffer, and then centrifuged the column. All of the non-protein substances in the cell were filtered out to the bottom of the column in the first few washes. This meant that by our fifth or six wash, the substances coming through the column were our protein, 2QRU. We then ran a gel with our protein extract to see if it was successfully expressed. What we found, as expected, was that the band at our protein weight got stronger as the purification process went on, meaning the sample from our first wash had many bands signifying other proteins in the cell, not just ours. But, the sample from our last wash had 1 clean band at the expected weight of our protein, 33.8 kda. [[Image:Screen Shot 2018-05-21 at 10.18.50.png|thumb||left|500pxl|Figure 1:This is our protein gel]] Figure 1 is an SDS Page Gel showing the molecular weight of the puri ed 2QRU from February. The ladder is in kDa. [Lane 8 – Ladder (5 uL), Lane 9 – Cell ex- tract (15 uL), Lane 10 – Flow through 6 (15 uL), Lane 11 – Wash 1 (15 uL), Lane 12 – Wash 3 (15 uL), Lane 13 – Elution 1 (14 uL), Lane 14 – Elution 2 (14 uL), Lane 15 – Elution 3 (14 uL)]<br />
</StructureSection><br />
== Structural Analysis ==<br />
[[Image:EfCD00584424 Map.png|thumb||left|500pxl|Figure 2:This is our plasmid visualized using Snap Gene]] Figure 2 shows what my protein looks like inside a plasmid. We used SnapGene to figure out the weight of our protein 33.8 kda. Once we proved that we could produce our protein, we started to think about how we wanted to characterize its functions. Since we know that function and structure go hand in hand, we also used a program called PyMol to first look at the 3D structure of our protein. Here is a visualization of my protein highlighting the <scene name='78/787197/1_2qru_cartoon/1'>alpha helices and beta sheets.</scene> It’s an alpha/beta hydrolase that has one chain. It's 816 base pairs long. Then, using Dali and pFam, we ran a search for other proteins that were structurally similar to ours. We found a few hits including 1TAH and 1C4X that are classified as esterases by aligning the active sites of these proteins to ours. [[Image:Screen Shot 2018-05-21 at 10.20.32.png|thumb||left|750pxl|Figure 3:These are our PyMol alignments]] The left most photo in Figure 3 shows the alignment of the active sites of 2QRU and 1TAH in PyMol. The RMS values of 1TAH’s catalytic triads compared to 2QRU’s catalytic triad was much lower than the RMS value of the full length alignment so this alignment is a better representation of the structural similarity. The middle photo showss the alignment between 2QRU and1C4X in PyMol. The RMS values of 1C4X’s catalytic triads compared to 2QRU’s catalytic triad was much lower than the RMS value of the full length alignment so this alignment is a better representation of the struc- tural similarity. The right most photo shows the alignment of 2QRU and 3FAK structures in PyMol.The RMS of 3FAK and 2QRU’s active site was much higher than the full structure alignment RMS, so this alignment is a better representation of the structural similarity.<br />
<br />
== Assays ==<br />
In order to determine if our protein was an esterase, we used an assay found in a published paper that sought to characterize 3FAK as an esterase. They performed a colorimetric assay which we modified for our study. The study we researched used p-nitrophenyl butyrate to test if 3FAK was an esterase. When the protein came into contact with this liquid, the entire solution turned yellow. We made a blank cuvette with Tris buffer and p-nitrophenyl butyrate. Then we added our protein and measured how the color of the solution changed over 30 sec intervals for 2 min. We repeated this using various concentrations of protein. <br />
<br />
== Results ==<br />
[[Image:Screen Shot 2018-05-21 at 10.20.12.png|thumb||left|1000pxl|Figure 4:This is a graph summerizing our assay results.]] Figure 4 shows the results of the successful Esterase Assay with varied concentrations of NPB dissolved in n-Heptane. The change in colorimeter absorbance over time is shown. We graphed the OD readings for each concentration and found that 0.375M of 2QRU produced the highest rate of reaction with p-nitrophenyl butyrate. We can first conclude that our protein is an esterase, but our results were slightly confusing. We expected that the highest concentration of PNB tested, 0.5M, would have the fastest reaction rate. However, we since the second highest concentration produced the highest reaction rate, we thought perhaps 0.375M could be the optimal concentration. <br />
Possible errors include inconsistent timing when inserting the induced cuvette into the colorimeter. This may explain why 0.375M of p-nitrophenyl butyrate had the steepest reaction rate with 2QRU even though we ran tests with higher concentrations of p-nitrophenyl butyrate. P-nitrophenyl butyrate concentrations below 0.15M failed to produce a measurable reaction with 2QRU. Thus, we tried the assay again with higher concentrations of p-nitrophenyl butyrate. Another error occurred at the beginning of our research to prove that 2QRU is an esterase. Another error occurred we performed an enzymatic assay of an esterase from Sigma Aldrich that tracked the enzymatic reaction by measuring the pH change over time. After attempting the assay, there were no results and therefore we could not characterize 2QRU. This lead us to our successful assay.<br />
<br />
== Future Directions ==<br />
After coming to the conclusion that 2QRU is an esterase, our next research activity would be to test if 2QRU is also a lipase. Three students from The Pingry School in New Jersey per- formed a lipase assay with 2QRU using nitrophenyl palmitate rather than p-nitrophenyl butyrate to determine if 2QRU is a lipase. Nitrophenyl palmitate has (CH2)14 side chain than nitrophenyl butyrate, so the protein has to act on a bigger substrate. If this assay works for us, then would be able to conclude that 2QRU is also a lipase. Another avenue to explore after con rming 2QRU is a lipase is mutagenesis. If we mutated the catalytic triad responsible for the reaction with nitrophenyl palmitate and ran the lipase assay again, we should see no reaction occur in the cuvette. We can use this to con rm that 2QRU is a lipase. The same thing can be done to confirm 2QRU is an esterase if we run the esterase assay using p-nitrophenyl butyrate.<br />
<br />
== References ==<br />
[1]Alberts, Bruce. Molecular Biology of the Cell. 6th ed. New York, NY: Garland Science, Taylor and Francis Group, 2015.<br />
[2] Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) “Basic local alignment search tool.” J. Mol. Biol. 215:403-410. PubMed.<br />
[3] “Enzymatic Assay of an Esterase.” Sigma-Aldrich. Accessed April 27, 2018. https://www.sigmaaldrich.com/technical-documents/protocols/biology/enzy- matic-assay-of-esterase.html.<br />
[4] Lisa Holm; Laura M. Laakso (2016) Dali server update. Nucleic acids research 44 (W1), W351-W355. PDF<br />
[5]“Milestones Tables.” PSI Structural Biology Knowledgebase. Accessed April 27, 2018. http://targetdb.pdb.org/Metrics/MilestonesTables.html.<br />
[6] Nam, Ki Hyun, Min-Young Kim, Soo-Jin Kim, Amit Pryadarshi, Won Ho Lee, and KwangYeon Hwang. “Structural and Functional Analysis of a Novel EstE5 Belonging to the Subfamily of Hormone-Sensitive Lipase.” Elsevier, December 29, 2008. https://www.ncbi.nlm.nih.gov/pubmed/19116143.<br />
[7] PDB: 2QRU Cuff, M.E., Volkart, L., Moy, S., Joachimiak, A.Structure of an alpha/beta hydrolase superfamily protein from Enterococcus faecalis.<br />
[8] ProMol Project. Dr. Paul Craig, Dr. Herbert Bernstein, Dr. Jeff Mills at Rochester Institute of Technology.<br />
[9] SnapGene Software (from GSL Biotech; available at snapgene.com).<br />
[10] The Pfam protein families database: towards a more sustainable future: R.D. Finn, P. Coggill, R.Y. Eberhardt, S.R. Eddy, J. Mistry, A.L. Mitchell, S.C. Potter, M. Punta, M. Qureshi, A. Sangrador-Vegas, G.A. Salazar, J. Tate, A. BatemanNucleic Acids Research (2016) Database Issue 44:D279-D285<br />
[11] The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.<br />
[12] Zhong, Qixin and Charles E. Glatz. “Enzymatic Assay Method for Evaluating the Lipase<br />
Activity in Complex Extracts from Transgenic Corn Seed.” Journal of Agricultural and Food Chemistry. 2006(54): 3181-3185. https://lib.dr.iastate.edu/cgi/view- content.cgi?referer=&httpsredir=1&article=1075&context=cbe_pubs.<br />
[13] Messaoudi, Abdelmonem & Belguith, Hatem & Gram, Imen & Ben Hamida, Jeannette. (2010). Classi cation of EC 3.1.1.3 bacterial true lipases using phyloge- netic analysis. African Journal of Biotechnology. 9. 8243-8247. 10.5897/AJB10.721.</div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_6&diff=2904803User:Jennifer Taylor/Sandbox 62018-05-26T21:13:39Z<p>Jennifer Taylor: </p>
<hr />
<div>== Overview ==<br />
Throughout the course of this past year, we attempted to determine the function of our protein 2QRU. Protein shape and protein function are really closely related, so if two proteins that look similar are compared, there is a high likelihood that they do the same thing. This was the underlying basis for our project. After initial computer analysis, we found that many of the proteins that had structural similarity to 2QRU were esterases. Thus, our preliminary hypothesis predicted that 2QRU would be a lipase, a subclass of esterases. We performed an esterase assay to prove that 2QRU was an esterase first, and are yet to determine if it can be classified as a lipase. [[Image: Screen_Shot_2018-05-21_at_10.20.55.png|thumb||left|800pxl|Figure 1:Diagrams Figure 1 (A-D clockwise) A: Hydrolases simply refer to any enzyme that catalyzes a hydrolysis reaction where H20 is added to cleave chemical bonds. Figure B: Esterases split esters into an alcohol and an acid through hydrolysis Figure C: Lipases catalyze the hydrolysis of lipids, or fats. Lipases are most commonly found on ∂/ß hydrolase chain and often contain the catalytic triad serine, histidine, and aspartic acid. Figure D: Lipid Chain with an extend- ed hydrocarbon chain, similar to the nitrophenyl palmitate.]]<br />
<br />
== Background ==<br />
Proteins are one of four major macromolecules in biology. Present in nearly every living organism, proteins have a diverse set of functions ranging from regulating cell activity to catalyze reactions. Due to the sheer number of proteins in existence, there still remain many to discover and characterize. In 2000, the Protein Structure Initiative began an attempt to solve 3D-structures of proteins with known sequences in order to understand their functions. Though the initiative was successful, they faced financial drawbacks in 2015. There still remain several protein structures with unknown functions in a public database called the Protein Data Bank. In a final PSI summary published in 2017, 6920 structures had been solved in their seventeen years of work. What we tried to do is take one of the protein structures solved by the PSI and characterize its function.<br />
<StructureSection load='2qru' size='340' side='right' caption='Caption for this structure' scene=''><br />
== Initial Process ==<br />
The first thing we did was try to see if we could express our protein in a cell. We first tried to insert our plasmid containing 2QRU into DH5∂ cells but found that this cell didn't produce the T7 polymerase that was necessary to transcribe 2QRU. We then switched to E. Coli cells. We inserted our plasmid into E. Coli using a standard transformation protocol. We then measured the concentration of our plasmid and then ran an SDS page gel to determine If our plasmid was successfully expressed in our cells. After that, we purified our protein or essentially squeezed it out of our cells to get protein concentrate. We did this using His-Pur Nickel-NTA Spin columns. We put our cell extract into a column, added a specific buffer, and then centrifuged the column. All of the non-protein substances in the cell were filtered out to the bottom of the column in the first few washes. This meant that by our fifth or six wash, the substances coming through the column were our protein, 2QRU. We then ran a gel with our protein extract to see if it was successfully expressed. What we found, as expected, was that the band at our protein weight got stronger as the purification process went on, meaning the sample from our first wash had many bands signifying other proteins in the cell, not just ours. But, the sample from our last wash had 1 clean band at the expected weight of our protein, 33.8 kda. [[Image:Screen Shot 2018-05-21 at 10.18.50.png|thumb||left|500pxl|Figure 1:This is our protein gel]] Figure 1 is an SDS Page Gel showing the molecular weight of the puri ed 2QRU from February. The ladder is in kDa. [Lane 8 – Ladder (5 uL), Lane 9 – Cell ex- tract (15 uL), Lane 10 – Flow through 6 (15 uL), Lane 11 – Wash 1 (15 uL), Lane 12 – Wash 3 (15 uL), Lane 13 – Elution 1 (14 uL), Lane 14 – Elution 2 (14 uL), Lane 15 – Elution 3 (14 uL)]<br />
</StructureSection><br />
== Structural Analysis ==<br />
[[Image:EfCD00584424 Map.png|thumb||left|500pxl|Figure 2:This is our plasmid visualized using Snap Gene]] Figure 2 shows what my protein looks like inside a plasmid. We used SnapGene to figure out the weight of our protein 33.8 kda. Once we proved that we could produce our protein, we started to think about how we wanted to characterize its functions. Since we know that function and structure go hand in hand, we also used a program called PyMol to first look at the 3D structure of our protein. Here is a visualization of my protein highlighting the <scene name='78/787197/1_2qru_cartoon/1'>alpha helices and beta sheets.</scene> It’s an alpha/beta hydrolase that has one chain. It's 816 base pairs long. Then, using Dali and pFam, we ran a search for other proteins that were structurally similar to ours. We found a few hits including 1TAH and 1C4X that are classified as esterases by aligning the active sites of these proteins to ours. [[Image:Screen Shot 2018-05-21 at 10.20.32.png|thumb||left|750pxl|Figure 3:These are our PyMol alignments]] The left most photo in Figure 3 shows the alignment of the active sites of 2QRU and 1TAH in PyMol. The RMS values of 1TAH’s catalytic triads compared to 2QRU’s catalytic triad was much lower than the RMS value of the full length alignment so this alignment is a better representation of the structural similarity. The middle photo showss the alignment between 2QRU and1C4X in PyMol. The RMS values of 1C4X’s catalytic triads compared to 2QRU’s catalytic triad was much lower than the RMS value of the full length alignment so this alignment is a better representation of the struc- tural similarity. The right most photo shows the alignment of 2QRU and 3FAK structures in PyMol.The RMS of 3FAK and 2QRU’s active site was much higher than the full structure alignment RMS, so this alignment is a better representation of the structural similarity.<br />
<br />
== Assays ==<br />
In order to determine if our protein was an esterase, we used an assay found in a published paper that sought to characterize 3FAK as an esterase. They performed a colorimetric assay which we modified for our study. The study we researched used p-nitrophenyl butyrate to test if 3FAK was an esterase. When the protein came into contact with this liquid, the entire solution turned yellow. We made a blank cuvette with Tris buffer and p-nitrophenyl butyrate. Then we added our protein and measured how the color of the solution changed over 30 sec intervals for 2 min. We repeated this using various concentrations of protein. <br />
<br />
== Results ==<br />
[[Image:Screen Shot 2018-05-21 at 10.20.12.png|thumb||left|1000pxl|Figure 4:This is a graph summerizing our assay results.]] Figure 4 shows the results of the successful Esterase Assay with varied concentrations of NPB dissolved in n-Heptane. The change in colorimeter absorbance over time is shown. We graphed the OD readings for each concentration and found that 0.375M of 2QRU produced the highest rate of reaction with p-nitrophenyl butyrate. We can first conclude that our protein is an esterase, but our results were slightly confusing. We expected that the highest concentration of PNB tested, 0.5M, would have the fastest reaction rate. However, we since the second highest concentration produced the highest reaction rate, we thought perhaps 0.375M could be the optimal concentration. <br />
Possible errors include inconsistent timing when inserting the induced cuvette into the colorimeter. This may explain why 0.375M of p-nitrophenyl butyrate had the steepest reaction rate with 2QRU even though we ran tests with higher concentrations of p-nitrophenyl butyrate. P-nitrophenyl butyrate concentrations below 0.15M failed to produce a measurable reaction with 2QRU. Thus, we tried the assay again with higher concentrations of p-nitrophenyl butyrate. Another error occurred at the beginning of our research to prove that 2QRU is an esterase. Another error occurred we performed an enzymatic assay of an esterase from Sigma Aldrich that tracked the enzymatic reaction by measuring the pH change over time. After attempting the assay, there were no results and therefore we could not characterize 2QRU. This lead us to our successful assay.<br />
<br />
== Future Directions ==<br />
After coming to the conclusion that 2QRU is an esterase, our next research activity would be to test if 2QRU is also a lipase. Three students from The Pingry School in New Jersey per- formed a lipase assay with 2QRU using nitrophenyl palmitate rather than p-nitrophenyl butyrate to determine if 2QRU is a lipase. Nitrophenyl palmitate has (CH2)14 side chain than nitrophenyl butyrate, so the protein has to act on a bigger substrate. If this assay works for us, then would be able to conclude that 2QRU is also a lipase. Another avenue to explore after con rming 2QRU is a lipase is mutagenesis. If we mutated the catalytic triad responsible for the reaction with nitrophenyl palmitate and ran the lipase assay again, we should see no reaction occur in the cuvette. We can use this to con rm that 2QRU is a lipase. The same thing can be done to confirm 2QRU is an esterase if we run the esterase assay using p-nitrophenyl butyrate.<br />
<br />
== References ==<br />
[1]Alberts, Bruce. Molecular Biology of the Cell. 6th ed. New York, NY: Garland Science, Taylor and Francis Group, 2015.<br />
[2] Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) “Basic local alignment search tool.” J. Mol. Biol. 215:403-410. PubMed.<br />
[3] “Enzymatic Assay of an Esterase.” Sigma-Aldrich. Accessed April 27, 2018. https://www.sigmaaldrich.com/technical-documents/protocols/biology/enzy- matic-assay-of-esterase.html.<br />
[4] Lisa Holm; Laura M. Laakso (2016) Dali server update. Nucleic acids research 44 (W1), W351-W355. PDF<br />
[5]“Milestones Tables.” PSI Structural Biology Knowledgebase. Accessed April 27, 2018. http://targetdb.pdb.org/Metrics/MilestonesTables.html.<br />
[6] Nam, Ki Hyun, Min-Young Kim, Soo-Jin Kim, Amit Pryadarshi, Won Ho Lee, and KwangYeon Hwang. “Structural and Functional Analysis of a Novel EstE5 Belonging to the Subfamily of Hormone-Sensitive Lipase.” Elsevier, December 29, 2008. https://www.ncbi.nlm.nih.gov/pubmed/19116143.<br />
[7] PDB: 2QRU Cuff, M.E., Volkart, L., Moy, S., Joachimiak, A.Structure of an alpha/beta hydrolase superfamily protein from Enterococcus faecalis.<br />
[8] ProMol Project. Dr. Paul Craig, Dr. Herbert Bernstein, Dr. Jeff Mills at Rochester Institute of Technology.<br />
[9] SnapGene Software (from GSL Biotech; available at snapgene.com).<br />
[10] The Pfam protein families database: towards a more sustainable future: R.D. Finn, P. Coggill, R.Y. Eberhardt, S.R. Eddy, J. Mistry, A.L. Mitchell, S.C. Potter, M. Punta, M. Qureshi, A. Sangrador-Vegas, G.A. Salazar, J. Tate, A. BatemanNucleic Acids Research (2016) Database Issue 44:D279-D285<br />
[11] The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.<br />
[12] Zhong, Qixin and Charles E. Glatz. “Enzymatic Assay Method for Evaluating the Lipase<br />
Activity in Complex Extracts from Transgenic Corn Seed.” Journal of Agricultural and Food Chemistry. 2006(54): 3181-3185. https://lib.dr.iastate.edu/cgi/view- content.cgi?referer=&httpsredir=1&article=1075&context=cbe_pubs.<br />
[13] Messaoudi, Abdelmonem & Belguith, Hatem & Gram, Imen & Ben Hamida, Jeannette. (2010). Classi cation of EC 3.1.1.3 bacterial true lipases using phyloge- netic analysis. African Journal of Biotechnology. 9. 8243-8247. 10.5897/AJB10.721.</div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_6&diff=2904802User:Jennifer Taylor/Sandbox 62018-05-26T21:13:08Z<p>Jennifer Taylor: </p>
<hr />
<div>== Overview ==<br />
Throughout the course of this past year, we attempted to determine the function of our protein 2QRU. Protein shape and protein function are really closely related, so if two proteins that look similar are compared, there is a high likelihood that they do the same thing. This was the underlying basis for our project. After initial computer analysis, we found that many of the proteins that had structural similarity to 2QRU were esterases. Thus, our preliminary hypothesis predicted that 2QRU would be a lipase, a subclass of esterases. We performed an esterase assay to prove that 2QRU was an esterase first, and are yet to determine if it can be classified as a lipase. [[Image: Screen_Shot_2018-05-21_at_10.20.55.png|thumb||left|500pxl|Figure 1:Diagrams Figure 1 (A-D clockwise) A: Hydrolases simply refer to any enzyme that catalyzes a hydrolysis reaction where H20 is added to cleave chemical bonds. Figure B: Esterases split esters into an alcohol and an acid through hydrolysis Figure C: Lipases catalyze the hydrolysis of lipids, or fats. Lipases are most commonly found on ∂/ß hydrolase chain and often contain the catalytic triad serine, histidine, and aspartic acid. Figure D: Lipid Chain with an extend- ed hydrocarbon chain, similar to the nitrophenyl palmitate.]]<br />
<br />
== Background ==<br />
Proteins are one of four major macromolecules in biology. Present in nearly every living organism, proteins have a diverse set of functions ranging from regulating cell activity to catalyze reactions. Due to the sheer number of proteins in existence, there still remain many to discover and characterize. In 2000, the Protein Structure Initiative began an attempt to solve 3D-structures of proteins with known sequences in order to understand their functions. Though the initiative was successful, they faced financial drawbacks in 2015. There still remain several protein structures with unknown functions in a public database called the Protein Data Bank. In a final PSI summary published in 2017, 6920 structures had been solved in their seventeen years of work. What we tried to do is take one of the protein structures solved by the PSI and characterize its function.<br />
<StructureSection load='2qru' size='340' side='right' caption='Caption for this structure' scene=''><br />
== Initial Process ==<br />
The first thing we did was try to see if we could express our protein in a cell. We first tried to insert our plasmid containing 2QRU into DH5∂ cells but found that this cell didn't produce the T7 polymerase that was necessary to transcribe 2QRU. We then switched to E. Coli cells. We inserted our plasmid into E. Coli using a standard transformation protocol. We then measured the concentration of our plasmid and then ran an SDS page gel to determine If our plasmid was successfully expressed in our cells. After that, we purified our protein or essentially squeezed it out of our cells to get protein concentrate. We did this using His-Pur Nickel-NTA Spin columns. We put our cell extract into a column, added a specific buffer, and then centrifuged the column. All of the non-protein substances in the cell were filtered out to the bottom of the column in the first few washes. This meant that by our fifth or six wash, the substances coming through the column were our protein, 2QRU. We then ran a gel with our protein extract to see if it was successfully expressed. What we found, as expected, was that the band at our protein weight got stronger as the purification process went on, meaning the sample from our first wash had many bands signifying other proteins in the cell, not just ours. But, the sample from our last wash had 1 clean band at the expected weight of our protein, 33.8 kda. [[Image:Screen Shot 2018-05-21 at 10.18.50.png|thumb||left|500pxl|Figure 1:This is our protein gel]] Figure 1 is an SDS Page Gel showing the molecular weight of the puri ed 2QRU from February. The ladder is in kDa. [Lane 8 – Ladder (5 uL), Lane 9 – Cell ex- tract (15 uL), Lane 10 – Flow through 6 (15 uL), Lane 11 – Wash 1 (15 uL), Lane 12 – Wash 3 (15 uL), Lane 13 – Elution 1 (14 uL), Lane 14 – Elution 2 (14 uL), Lane 15 – Elution 3 (14 uL)]<br />
</StructureSection><br />
== Structural Analysis ==<br />
[[Image:EfCD00584424 Map.png|thumb||left|500pxl|Figure 2:This is our plasmid visualized using Snap Gene]] Figure 2 shows what my protein looks like inside a plasmid. We used SnapGene to figure out the weight of our protein 33.8 kda. Once we proved that we could produce our protein, we started to think about how we wanted to characterize its functions. Since we know that function and structure go hand in hand, we also used a program called PyMol to first look at the 3D structure of our protein. Here is a visualization of my protein highlighting the <scene name='78/787197/1_2qru_cartoon/1'>alpha helices and beta sheets.</scene> It’s an alpha/beta hydrolase that has one chain. It's 816 base pairs long. Then, using Dali and pFam, we ran a search for other proteins that were structurally similar to ours. We found a few hits including 1TAH and 1C4X that are classified as esterases by aligning the active sites of these proteins to ours. [[Image:Screen Shot 2018-05-21 at 10.20.32.png|thumb||left|750pxl|Figure 3:These are our PyMol alignments]] The left most photo in Figure 3 shows the alignment of the active sites of 2QRU and 1TAH in PyMol. The RMS values of 1TAH’s catalytic triads compared to 2QRU’s catalytic triad was much lower than the RMS value of the full length alignment so this alignment is a better representation of the structural similarity. The middle photo showss the alignment between 2QRU and1C4X in PyMol. The RMS values of 1C4X’s catalytic triads compared to 2QRU’s catalytic triad was much lower than the RMS value of the full length alignment so this alignment is a better representation of the struc- tural similarity. The right most photo shows the alignment of 2QRU and 3FAK structures in PyMol.The RMS of 3FAK and 2QRU’s active site was much higher than the full structure alignment RMS, so this alignment is a better representation of the structural similarity.<br />
<br />
== Assays ==<br />
In order to determine if our protein was an esterase, we used an assay found in a published paper that sought to characterize 3FAK as an esterase. They performed a colorimetric assay which we modified for our study. The study we researched used p-nitrophenyl butyrate to test if 3FAK was an esterase. When the protein came into contact with this liquid, the entire solution turned yellow. We made a blank cuvette with Tris buffer and p-nitrophenyl butyrate. Then we added our protein and measured how the color of the solution changed over 30 sec intervals for 2 min. We repeated this using various concentrations of protein. <br />
<br />
== Results ==<br />
[[Image:Screen Shot 2018-05-21 at 10.20.12.png|thumb||left|1000pxl|Figure 4:This is a graph summerizing our assay results.]] Figure 4 shows the results of the successful Esterase Assay with varied concentrations of NPB dissolved in n-Heptane. The change in colorimeter absorbance over time is shown. We graphed the OD readings for each concentration and found that 0.375M of 2QRU produced the highest rate of reaction with p-nitrophenyl butyrate. We can first conclude that our protein is an esterase, but our results were slightly confusing. We expected that the highest concentration of PNB tested, 0.5M, would have the fastest reaction rate. However, we since the second highest concentration produced the highest reaction rate, we thought perhaps 0.375M could be the optimal concentration. <br />
Possible errors include inconsistent timing when inserting the induced cuvette into the colorimeter. This may explain why 0.375M of p-nitrophenyl butyrate had the steepest reaction rate with 2QRU even though we ran tests with higher concentrations of p-nitrophenyl butyrate. P-nitrophenyl butyrate concentrations below 0.15M failed to produce a measurable reaction with 2QRU. Thus, we tried the assay again with higher concentrations of p-nitrophenyl butyrate. Another error occurred at the beginning of our research to prove that 2QRU is an esterase. Another error occurred we performed an enzymatic assay of an esterase from Sigma Aldrich that tracked the enzymatic reaction by measuring the pH change over time. After attempting the assay, there were no results and therefore we could not characterize 2QRU. This lead us to our successful assay.<br />
<br />
== Future Directions ==<br />
After coming to the conclusion that 2QRU is an esterase, our next research activity would be to test if 2QRU is also a lipase. Three students from The Pingry School in New Jersey per- formed a lipase assay with 2QRU using nitrophenyl palmitate rather than p-nitrophenyl butyrate to determine if 2QRU is a lipase. Nitrophenyl palmitate has (CH2)14 side chain than nitrophenyl butyrate, so the protein has to act on a bigger substrate. If this assay works for us, then would be able to conclude that 2QRU is also a lipase. Another avenue to explore after con rming 2QRU is a lipase is mutagenesis. If we mutated the catalytic triad responsible for the reaction with nitrophenyl palmitate and ran the lipase assay again, we should see no reaction occur in the cuvette. We can use this to con rm that 2QRU is a lipase. The same thing can be done to confirm 2QRU is an esterase if we run the esterase assay using p-nitrophenyl butyrate.<br />
<br />
== References ==<br />
[1]Alberts, Bruce. Molecular Biology of the Cell. 6th ed. New York, NY: Garland Science, Taylor and Francis Group, 2015.<br />
[2] Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) “Basic local alignment search tool.” J. Mol. Biol. 215:403-410. PubMed.<br />
[3] “Enzymatic Assay of an Esterase.” Sigma-Aldrich. Accessed April 27, 2018. https://www.sigmaaldrich.com/technical-documents/protocols/biology/enzy- matic-assay-of-esterase.html.<br />
[4] Lisa Holm; Laura M. Laakso (2016) Dali server update. Nucleic acids research 44 (W1), W351-W355. PDF<br />
[5]“Milestones Tables.” PSI Structural Biology Knowledgebase. Accessed April 27, 2018. http://targetdb.pdb.org/Metrics/MilestonesTables.html.<br />
[6] Nam, Ki Hyun, Min-Young Kim, Soo-Jin Kim, Amit Pryadarshi, Won Ho Lee, and KwangYeon Hwang. “Structural and Functional Analysis of a Novel EstE5 Belonging to the Subfamily of Hormone-Sensitive Lipase.” Elsevier, December 29, 2008. https://www.ncbi.nlm.nih.gov/pubmed/19116143.<br />
[7] PDB: 2QRU Cuff, M.E., Volkart, L., Moy, S., Joachimiak, A.Structure of an alpha/beta hydrolase superfamily protein from Enterococcus faecalis.<br />
[8] ProMol Project. Dr. Paul Craig, Dr. Herbert Bernstein, Dr. Jeff Mills at Rochester Institute of Technology.<br />
[9] SnapGene Software (from GSL Biotech; available at snapgene.com).<br />
[10] The Pfam protein families database: towards a more sustainable future: R.D. Finn, P. Coggill, R.Y. Eberhardt, S.R. Eddy, J. Mistry, A.L. Mitchell, S.C. Potter, M. Punta, M. Qureshi, A. Sangrador-Vegas, G.A. Salazar, J. Tate, A. BatemanNucleic Acids Research (2016) Database Issue 44:D279-D285<br />
[11] The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.<br />
[12] Zhong, Qixin and Charles E. Glatz. “Enzymatic Assay Method for Evaluating the Lipase<br />
Activity in Complex Extracts from Transgenic Corn Seed.” Journal of Agricultural and Food Chemistry. 2006(54): 3181-3185. https://lib.dr.iastate.edu/cgi/view- content.cgi?referer=&httpsredir=1&article=1075&context=cbe_pubs.<br />
[13] Messaoudi, Abdelmonem & Belguith, Hatem & Gram, Imen & Ben Hamida, Jeannette. (2010). Classi cation of EC 3.1.1.3 bacterial true lipases using phyloge- netic analysis. African Journal of Biotechnology. 9. 8243-8247. 10.5897/AJB10.721.</div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_6&diff=2904801User:Jennifer Taylor/Sandbox 62018-05-26T21:11:22Z<p>Jennifer Taylor: </p>
<hr />
<div>== Overview ==<br />
Throughout the course of this past year, we attempted to determine the function of our protein 2QRU. Protein shape and protein function are really closely related, so if two proteins that look similar are compared, there is a high likelihood that they do the same thing. This was the underlying basis for our project. After initial computer analysis, we found that many of the proteins that had structural similarity to 2QRU were esterases. Thus, our preliminary hypothesis predicted that 2QRU would be a lipase, a subclass of esterases. We performed an esterase assay to prove that 2QRU was an esterase first, and are yet to determine if it can be classified as a lipase. [[Image:Screen Shot 2018-05-21 at 10.20.55|thumb||left|500pxl|Figure 1:Diagrams Figure 1 (A-D clockwise) A: Hydrolases simply refer to any enzyme that catalyzes a hydrolysis reaction where H20 is added to cleave chemical bonds. Figure B: Esterases split esters into an alcohol and an acid through hydrolysis Figure C: Lipases catalyze the hydrolysis of lipids, or fats. Lipases are most commonly found on ∂/ß hydrolase chain and often contain the catalytic triad serine, histidine, and aspartic acid. Figure D: Lipid Chain with an extend- ed hydrocarbon chain, similar to the nitrophenyl palmitate.]]<br />
<br />
== Background ==<br />
Proteins are one of four major macromolecules in biology. Present in nearly every living organism, proteins have a diverse set of functions ranging from regulating cell activity to catalyze reactions. Due to the sheer number of proteins in existence, there still remain many to discover and characterize. In 2000, the Protein Structure Initiative began an attempt to solve 3D-structures of proteins with known sequences in order to understand their functions. Though the initiative was successful, they faced financial drawbacks in 2015. There still remain several protein structures with unknown functions in a public database called the Protein Data Bank. In a final PSI summary published in 2017, 6920 structures had been solved in their seventeen years of work. What we tried to do is take one of the protein structures solved by the PSI and characterize its function.<br />
<StructureSection load='2qru' size='340' side='right' caption='Caption for this structure' scene=''><br />
== Initial Process ==<br />
The first thing we did was try to see if we could express our protein in a cell. We first tried to insert our plasmid containing 2QRU into DH5∂ cells but found that this cell didn't produce the T7 polymerase that was necessary to transcribe 2QRU. We then switched to E. Coli cells. We inserted our plasmid into E. Coli using a standard transformation protocol. We then measured the concentration of our plasmid and then ran an SDS page gel to determine If our plasmid was successfully expressed in our cells. After that, we purified our protein or essentially squeezed it out of our cells to get protein concentrate. We did this using His-Pur Nickel-NTA Spin columns. We put our cell extract into a column, added a specific buffer, and then centrifuged the column. All of the non-protein substances in the cell were filtered out to the bottom of the column in the first few washes. This meant that by our fifth or six wash, the substances coming through the column were our protein, 2QRU. We then ran a gel with our protein extract to see if it was successfully expressed. What we found, as expected, was that the band at our protein weight got stronger as the purification process went on, meaning the sample from our first wash had many bands signifying other proteins in the cell, not just ours. But, the sample from our last wash had 1 clean band at the expected weight of our protein, 33.8 kda. [[Image:Screen Shot 2018-05-21 at 10.18.50.png|thumb||left|500pxl|Figure 1:This is our protein gel]] Figure 1 is an SDS Page Gel showing the molecular weight of the puri ed 2QRU from February. The ladder is in kDa. [Lane 8 – Ladder (5 uL), Lane 9 – Cell ex- tract (15 uL), Lane 10 – Flow through 6 (15 uL), Lane 11 – Wash 1 (15 uL), Lane 12 – Wash 3 (15 uL), Lane 13 – Elution 1 (14 uL), Lane 14 – Elution 2 (14 uL), Lane 15 – Elution 3 (14 uL)]<br />
</StructureSection><br />
== Structural Analysis ==<br />
[[Image:EfCD00584424 Map.png|thumb||left|500pxl|Figure 2:This is our plasmid visualized using Snap Gene]] Figure 2 shows what my protein looks like inside a plasmid. We used SnapGene to figure out the weight of our protein 33.8 kda. Once we proved that we could produce our protein, we started to think about how we wanted to characterize its functions. Since we know that function and structure go hand in hand, we also used a program called PyMol to first look at the 3D structure of our protein. Here is a visualization of my protein highlighting the <scene name='78/787197/1_2qru_cartoon/1'>alpha helices and beta sheets.</scene> It’s an alpha/beta hydrolase that has one chain. It's 816 base pairs long. Then, using Dali and pFam, we ran a search for other proteins that were structurally similar to ours. We found a few hits including 1TAH and 1C4X that are classified as esterases by aligning the active sites of these proteins to ours. [[Image:Screen Shot 2018-05-21 at 10.20.32.png|thumb||left|750pxl|Figure 3:These are our PyMol alignments]] The left most photo in Figure 3 shows the alignment of the active sites of 2QRU and 1TAH in PyMol. The RMS values of 1TAH’s catalytic triads compared to 2QRU’s catalytic triad was much lower than the RMS value of the full length alignment so this alignment is a better representation of the structural similarity. The middle photo showss the alignment between 2QRU and1C4X in PyMol. The RMS values of 1C4X’s catalytic triads compared to 2QRU’s catalytic triad was much lower than the RMS value of the full length alignment so this alignment is a better representation of the struc- tural similarity. The right most photo shows the alignment of 2QRU and 3FAK structures in PyMol.The RMS of 3FAK and 2QRU’s active site was much higher than the full structure alignment RMS, so this alignment is a better representation of the structural similarity.<br />
<br />
== Assays ==<br />
In order to determine if our protein was an esterase, we used an assay found in a published paper that sought to characterize 3FAK as an esterase. They performed a colorimetric assay which we modified for our study. The study we researched used p-nitrophenyl butyrate to test if 3FAK was an esterase. When the protein came into contact with this liquid, the entire solution turned yellow. We made a blank cuvette with Tris buffer and p-nitrophenyl butyrate. Then we added our protein and measured how the color of the solution changed over 30 sec intervals for 2 min. We repeated this using various concentrations of protein. <br />
<br />
== Results ==<br />
[[Image:Screen Shot 2018-05-21 at 10.20.12.png|thumb||left|1000pxl|Figure 4:This is a graph summerizing our assay results.]] Figure 4 shows the results of the successful Esterase Assay with varied concentrations of NPB dissolved in n-Heptane. The change in colorimeter absorbance over time is shown. We graphed the OD readings for each concentration and found that 0.375M of 2QRU produced the highest rate of reaction with p-nitrophenyl butyrate. We can first conclude that our protein is an esterase, but our results were slightly confusing. We expected that the highest concentration of PNB tested, 0.5M, would have the fastest reaction rate. However, we since the second highest concentration produced the highest reaction rate, we thought perhaps 0.375M could be the optimal concentration. <br />
Possible errors include inconsistent timing when inserting the induced cuvette into the colorimeter. This may explain why 0.375M of p-nitrophenyl butyrate had the steepest reaction rate with 2QRU even though we ran tests with higher concentrations of p-nitrophenyl butyrate. P-nitrophenyl butyrate concentrations below 0.15M failed to produce a measurable reaction with 2QRU. Thus, we tried the assay again with higher concentrations of p-nitrophenyl butyrate. Another error occurred at the beginning of our research to prove that 2QRU is an esterase. Another error occurred we performed an enzymatic assay of an esterase from Sigma Aldrich that tracked the enzymatic reaction by measuring the pH change over time. After attempting the assay, there were no results and therefore we could not characterize 2QRU. This lead us to our successful assay.<br />
<br />
== Future Directions ==<br />
After coming to the conclusion that 2QRU is an esterase, our next research activity would be to test if 2QRU is also a lipase. Three students from The Pingry School in New Jersey per- formed a lipase assay with 2QRU using nitrophenyl palmitate rather than p-nitrophenyl butyrate to determine if 2QRU is a lipase. Nitrophenyl palmitate has (CH2)14 side chain than nitrophenyl butyrate, so the protein has to act on a bigger substrate. If this assay works for us, then would be able to conclude that 2QRU is also a lipase. Another avenue to explore after con rming 2QRU is a lipase is mutagenesis. If we mutated the catalytic triad responsible for the reaction with nitrophenyl palmitate and ran the lipase assay again, we should see no reaction occur in the cuvette. We can use this to con rm that 2QRU is a lipase. The same thing can be done to confirm 2QRU is an esterase if we run the esterase assay using p-nitrophenyl butyrate.<br />
<br />
== References ==<br />
[1]Alberts, Bruce. Molecular Biology of the Cell. 6th ed. New York, NY: Garland Science, Taylor and Francis Group, 2015.<br />
[2] Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) “Basic local alignment search tool.” J. Mol. Biol. 215:403-410. PubMed.<br />
[3] “Enzymatic Assay of an Esterase.” Sigma-Aldrich. Accessed April 27, 2018. https://www.sigmaaldrich.com/technical-documents/protocols/biology/enzy- matic-assay-of-esterase.html.<br />
[4] Lisa Holm; Laura M. Laakso (2016) Dali server update. Nucleic acids research 44 (W1), W351-W355. PDF<br />
[5]“Milestones Tables.” PSI Structural Biology Knowledgebase. Accessed April 27, 2018. http://targetdb.pdb.org/Metrics/MilestonesTables.html.<br />
[6] Nam, Ki Hyun, Min-Young Kim, Soo-Jin Kim, Amit Pryadarshi, Won Ho Lee, and KwangYeon Hwang. “Structural and Functional Analysis of a Novel EstE5 Belonging to the Subfamily of Hormone-Sensitive Lipase.” Elsevier, December 29, 2008. https://www.ncbi.nlm.nih.gov/pubmed/19116143.<br />
[7] PDB: 2QRU Cuff, M.E., Volkart, L., Moy, S., Joachimiak, A.Structure of an alpha/beta hydrolase superfamily protein from Enterococcus faecalis.<br />
[8] ProMol Project. Dr. Paul Craig, Dr. Herbert Bernstein, Dr. Jeff Mills at Rochester Institute of Technology.<br />
[9] SnapGene Software (from GSL Biotech; available at snapgene.com).<br />
[10] The Pfam protein families database: towards a more sustainable future: R.D. Finn, P. Coggill, R.Y. Eberhardt, S.R. Eddy, J. Mistry, A.L. Mitchell, S.C. Potter, M. Punta, M. Qureshi, A. Sangrador-Vegas, G.A. Salazar, J. Tate, A. BatemanNucleic Acids Research (2016) Database Issue 44:D279-D285<br />
[11] The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.<br />
[12] Zhong, Qixin and Charles E. Glatz. “Enzymatic Assay Method for Evaluating the Lipase<br />
Activity in Complex Extracts from Transgenic Corn Seed.” Journal of Agricultural and Food Chemistry. 2006(54): 3181-3185. https://lib.dr.iastate.edu/cgi/view- content.cgi?referer=&httpsredir=1&article=1075&context=cbe_pubs.<br />
[13] Messaoudi, Abdelmonem & Belguith, Hatem & Gram, Imen & Ben Hamida, Jeannette. (2010). Classi cation of EC 3.1.1.3 bacterial true lipases using phyloge- netic analysis. African Journal of Biotechnology. 9. 8243-8247. 10.5897/AJB10.721.</div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_3&diff=2904793User:Jennifer Taylor/Sandbox 32018-05-25T14:05:10Z<p>Jennifer Taylor: </p>
<hr />
<div>== 3H04 Test Page==<br />
<br />
<StructureSection load='3H04' size='340' side='right' caption='Here is a 3D image of my Protein=''><br />
<br />
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br />
<br />
==Introduction==<br />
Beginning in 2000, the National Institutes of Health’s Protein Structure Initiative was a fifteen-year program that worked to solve the structures of many proteins with known DNA sequences. The present study analyzes one such protein, known as <scene name='78/787193/3h04_n_to_c_rainbow/1'>3H04</scene>, a predicted alpha/beta hydrolase found in Escherichia Coli. The specific objective of this study is to functionally characterize 3H04 based on structural analysis of the protein. ''In silico'' enzyme characterization for 3H04 was carried out using BLAST, Pfam, and Dali servers, followed by active site analysis using the PyMOL plug-in ProMOL. This structural and protein sequence analysis suggests PDB entries <scene name='78/787193/1azw/1'>1AZW</scene> and <scene name='78/787193/1ysc/1'>1YSC</scene> , respectively, are most similar to 3H04; however 1TAH and 3H04 share the most catalytic triad similarity. To investigate the lipase activity of 3H04 ''in vitro'', recombinant his-tagged protein was isolated from E. coli. <br />
<br />
Proteins are macromolecules that are present in nearly every earthly organism and represent a vast array of functions. Scientists have been able to structurally solve proteins at a faster rate than they have been able to identify the functions of these same proteins. In an effort to close this gap, the National Institutes of Health launched the Protein Structure Initiative, which was a fifteen-year program, starting in 2000, that worked to solve the structures of many proteins with known DNA sequences. However, there are still hundreds proteins housed within the Protein Data Bank that have unidentified functions. Researchers are continuing to advocate for further analysis of these proteins through the development of BASIL modules that explain how to use ProMOL to analyzes the structural similarities between the active sites of proteins.<br />
<br />
== Overview ==<br />
This project seeks to determine the function of a protein with an identified structure. The query protein, in this case, can be identified by the Protein Data Bank Identification (PDB ID) code <scene name='78/787193/3h04_n_to_c_rainbow/1'>3H04</scene>. 3H04 is a alpha/beta hydrolase found in Escherichia Coli, a bacterium that resides in the lower intestines of warm-blooded mammals. The more specific purpose of this study is to characterize the function of 3H04. Plasmid purification is just one method for identifying an unidentified protein. Protein purification in bacterial cells requires the plasmids, a small circular double-stranded DNA molecule, to be purified and identified. The purification of the plasmid is meant to isolate and purify the plasmid DNA. The protein in the plasmid is identified through bacterial cells, and grows against ampicillin so the bacterial cells are growing against the antibiotic; the cells that grow have AmpR (Ampicillin Resistance gene). And in efforts to characterize our protein, 3H04, we are comparing it to other proteins with significant parts in common to hopefully help characterize the function of 3H04. The comparison of the different aspects of our protein to different aspects of other proteins also helped us determine our assay.<br />
<br />
==Hypothesis==<br />
In the beginning, we predicted that our protein was an esterase, because originally our most promising homologs were an aminopeptidase and an esterase. And even though the most hits from our Promol search were enzyme class 3.4 (aminopeptidases), no further information was found about the catalytic triads of these hits. We decided to use a homolog in E.C. 3.1 (esterase) because there was information about their catalytic triads. We knew that our protein is a hydrolase, and we believe that 3H04 is characterized as an esterase (3.1), instead of an aminopeptidase (3.4). Even though the 3.4 category proteins had the most motif structure similarity in Promol, we chose <scene name='78/787193/1tah/1'>1TAH</scene> (a lipase) as our target homolog. 1TAH is a characterized protein and has a similar catalytic triad to what we predict for our protein (3H04). When we aligned the two catalytic triads <scene name='78/787193/Catalytic_triad_of_1tah/2'>1TAH</scene> and <scene name='78/787193/Catalytic_triad_3h04/1'>3H04</scene>, they had an RMS of 0.016. In the beginning we were looking for a homolog with more protein sequence and structure similarity, but because neither protein sequence or structure determine the function of 3H04, we decided that those two factors matter much less than the catalytic triad of the protein. So, we used Promol to find what the catalytic triad of 3H04 is and we used 1TAH as our target homolog for the catalytic triad. We predict, because the RMS was so low, that 3H04 and 1TAH will be having very similar enzymatic functions.<br />
<br />
== Methods ==<br />
<br />
We performed a Lipase Assay to determine what kind of esterase our protein is, because our target homolog (1TAH) is a lipase. The literature about 1TAH had no useful information about assays, but we used an assay from a paper regarding a different lipase. We knew that our protein was a hydrolase, and we believed it to be an esterase, and with this lipase assay we were able to deduce that our protein was an esterase and a lipase. To determine if 3H04 functions as a lipase on the p-Nitrophenyl Butyrate (pNPB), a colorimetric assay using a spectrophotometry protocol. After mixing pNPB in 2-propanol, add 50 mM Tris-HCl buffer (pH 8.0, containing 0.4% w/v Triton XI00 and 0.1% w/v arabic gum) to the mixture. To initiate reaction, add pNPB solution and measure absorbance using a UV spectrophotometer at 410 nm for 5-10 min, recording absorbance values at 20 second intervals. <br />
<br />
== Results ==<br />
<br />
<br />
[[Image:Lipase Assay OH.jpg|thumb|left|250px|Figure 1: Lipase Assay Result. 3H04 was tested with two different concentrations of pNPB to test how the concentration altered the rate of the reaction. ]] <br />
<br />
<br />
[[Image:Coomassie Gel OH.jpg|thumb|left|250px|Figure 2: Coomassie Gel. Coomassie Gel of 3H04 (Lanes 1-8). 1. Cell Extract, 2. Flow Through, 3. Wash 1, 4. Wash 3, 5. Elution 1, 6. Elution 2, 7. Elution 3, 8. Ladder. The protein is being expressed wherever there is a darker, thinker band in the same row as the 75 kDa.]] <br />
<br />
== Conclusion ==<br />
<br />
We've concluded that our protein is a lipase, and because we expressed our protein through E.coli, we believe that 3H04 functions in the lower intestine of warm blooded organisms. The blue dots in Figure 1 represent the pNPB at a lower concentration than the red dots. The blue dots represent the reaction happening at 0.15M and the red dots represent the reaction with a concentration of 0.5M, both concentrations showed positive results of a reaction, however the expected curve of the graph was not as steep as predicted with the original concentration of 0.15M. The concentration was increased to increase the curve of the graph so that the expected curve was attained. The stained coomaise gel in Figure 2 shows that our protein was being expressed through the bacterial cells, around the correct expected molecular weight. This protein, expressed, purified, and now characterized is in the enzyme class lipase.<br />
<br />
<br />
</StructureSection><br />
== References ==<br />
Zhang, R., Tesar, C., Sather, A., Clancy, S., Joachimiak, A., Midwest Center for Structural Genomics (MCSG)The crystal structure of the protein with unknown function from Staphylococcus aureus subsp. aureus Mu50<br />
The Pfam protein families database: towards a more sustainable future <https://nar.oxfordjournals.org/content/44/D1/D279.long>: R.D. Finn, P. Coggill, R.Y. Eberhardt, S.R. Eddy, J. Mistry, A.L. Mitchell, S.C. Potter, M. Punta, M. Qureshi, A. Sangrador-Vegas, G.A. Salazar, J. Tate, A. Bateman<br />
Nucleic Acids Research (2016) Database Issue 44:D279-D285<br />
PyMOL<br />
The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.</div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_1&diff=2904792User:Jennifer Taylor/Sandbox 12018-05-25T12:53:10Z<p>Jennifer Taylor: </p>
<hr />
<div>==YxiM from ''Bacillus subtilis''==<br />
<br />
<StructureSection load='2o14' size='340' side='right' caption='Structure of YxiM' scene=''><br />
<br />
== Background ==<br />
<br />
Proteins are an important type of macromolecule in biological systems and can be considered a sequence of subunits known as amino acids. The development of high-throughput genome squencing techniques allowed proteins to be sequenced more quickly than their structures could be solved. In an effort to close this gap, in 2000, the National Institutes of Health launched the 15-year Protein Structure Initiative. Many structures were deposited in the Protein Data Bank, but many of these proteins with solved structures, such as YxiM ([https://www.rcsb.org/structure/2o14 PDB ID: 2O14]), remain functionally uncharacterized. YxiM is transcribed by the ''yxiM'' gene from ''Bacillus subtilis'', a ubiquitous bacterial species that dwells in soil and gastrointestinal tracts. <scene name='78/787191/2o14_rainbow/2'>YxiM</scene> is 375 amino acids in length and its molecular weight is 41.8 kDa. It appears to have two domains: <scene name='78/787191/2o14_alpha/1'>one dominated by α-helices</scene>, and <scene name='78/787191/2o14_beta/1'>one by β-sheets</scene>.<br />
<br />
== ''In silico'' Analysis ==<br />
<br />
A common theme in biology is that form follows function. Thus, we used computer programs to find which proteins were most homologous to YxiM in terms of sequence and structure, with the expectation that YxiM is likely to be functionally similar to those proteins that have similar sequences and structures.<br />
<br />
[[Image:YxiM_green-1J00_blue.png|thumb|left|250px|'''Figure 1'''. YxiM (green) aligned with 1J00 (blue), RMSD = 2.692.]]<br />
<br />
We used BLAST and PFam to find characterized proteins whose sequences aligned best with YxiM. Sequence analysis suggests that YxiM is a GDSL-like lipase, a type of esterase. Esterases are molecules that hydrolyze (decompose) a class of organic molecules known as esters. GDSL-like lipases demonstrate broad substrate specificity due to their flexible structures. BLAST showed that the proteins 1J00, 1IVN, and 1JRL have the highest sequence homology to YxiM. These proteins are multifunctional hydrolases that show both esterase and protease activity.<br />
<br />
Next, we used PyMOL to align the 3D structures of the BLAST hits with that of YxiM. The proteins 1J00, 1IVN, and 1JRL all align well with the α-helix domain of YxiM.<br />
<br />
[[Image:YxiM-1BWR_Catalytic.png|thumb|right|250px|'''Figure 2'''. Active site of 1BWR aligned with the putative active site of YxiM, RMSD = 0.506.]]<br />
<br />
The Dali server finds the most similar proteins based on 3D structures, and the top 30 hits for YxiM were are all rhamnogalacturonan acetylesterases, GDSL lipases, LAE5s (hydrolases), or acetyl xylan esterases, which further suggests that YxiM is an esterase.<br />
<br />
Finally, we used ProMOL to perform a structural alignment of active sites of other proteins with YxiM to predict the active site of YxiM. We found that YxiM aligns best with the active site of IBWR, which is an esterase. The <scene name='78/787191/2o14_active_site/2'>putative catalytic triad</scene> of YxiM consists of amino acids S171, D339, and H342.<br />
<br />
Based on these analyses, we predicted that YxiM is an esterase and proceeded to perform ''in vitro'' assays to confirm esterase activity.<br />
<br />
== Plasmid Purification ==<br />
<br />
[[Image:PET21-YxiM_Map.png|thumb|right|250px|'''Figure 3'''. Plasmid map of pET21-''yxiM''.]]<br />
<br />
In order to study the protein YxiM, we ordered a plasmid that contains the gene that transcribes the protein. A plasmid is a type of circular bacterial DNA. By transforming (inserting) this plasmid (pET21-''yxiM'') into the bacteria (DH5α Competent ''E. coli''), we can use the bacteria to create more of the plasmid. Then, we performed a DNA miniprep to purify the plasmid for later use.<br />
<br />
== Bacterial Transformation ==<br />
<br />
While DH5α ''E. coli'' are good for purifying plasmids, BL21(DE3) ''E. coli'' are more efficient for expressing protein. Thus, we transformed the plasmid into BL21(DE3) for the purposes of protein expression. We plated the bacteria on agar with the antibiotic ampicillin. While normal ''E. coli'' will die in the presence of ampicillin, the pET21-''yxiM'' plasmid has a gene that allows bacteria to become ampicillin resistant. Thus, only bacteria that were successfully transformed by the plasmid will survive on the ampicillin plate, allowing us to select for bacteria that have been transformed and thus bacteria that will express the protein YxiM.<br />
<br />
== Protein Expression ==<br />
<br />
After a day, colonies of transformed bacteria were visible on the agar plates. To express YxiM, we inoculated a single colony of bacteria into a liquid culture. In the plasmid, the ''yxiM'' gene is under control of the ''lac'' operon. This means that in the absence of an inducer, the transcription of the ''yxiM'' gene is repressed. Thus, we added IPTG, to activate the operon and drive the transcription of the protein YxiM.<br />
<br />
== Protein Purification ==<br />
<br />
After several hours, the bacteria have produced a relatively large amount of YxiM. To collect the protein, which at this point remained inside the cells, the bacteria were lysed (their cell walls were burst). The resulting mixture consisted of various cellular proteins and debris. The plasmid DNA sequence that coded for YxiM added a "tag" of histidines at the end of the protein. This allows us to separate YxiM proteins from the other types of proteins in the ''E. coli'' cells because when the mixture is passed through a nickel column, the tagged YxiM proteins stick to the column, while the other proteins flow through. Finally, we added elution buffer to the columns, which caused the proteins to detach from the nickel columns, creating a solution of just the YxiM proteins.<br />
<br />
== Esterase Activity Assay ==<br />
<br />
[[Image:YxiM_Lineweaver-Burk_Plot.png|thumb|left|250px|'''Figure 4'''. Lineweaver-Burk plot of YxiM esterase activity.]]<br />
<br />
Now that we had purified protein, we could test the function of YxiM ''in vitro''. Since we believed that YxiM was an ester, we placed it in a buffered solution with 4-nitrophenyl butyrate, a type of ester. Esterases should hydrolyze 4-nitrophenyl butyrate, causing the products butyric acid and 4-nitrophenol to form. Since 4-nitrophenyl is a yellow color, the absorbance of the solution changes as more products are formed. We used colorimeter to measure the absorbance at 430 nm as a proxy for esterase activity. We found that the absorbance increases over time, which suggests that YxiM is indeed an esterase.<br />
<br />
Specifically, we found that the Lineweaver-Burk plot of esterase activity is linear. This is typical of enzymes, as predicted by the Michaelis-Menten model of enzyme kinetics. Technically, we did not construct a true Lineweaver-Burk plot, as we used absorbance as a proxy for molar concentration, but absorbance varies linearly with concentration, as shown by the Beer-Lambert law.<br />
<br />
== Discussion ==<br />
<br />
YxiM is a previously uncharacterized protein whose crystal structure has been solved and deposited in the PDB.<br />
<br />
Protein sequence analysis with BLAST shows that YxiM is likely an esterase. PyMOL shows that the top BLAST hits also align well with the 3D structure of the α-helix domain of YxiM. Almost all the top structural hits in Dali are esterases as well, and ProMOL shows that the active site of YxiM most resembles one of an esterase or protease. The same catalytic triad (S171, D339, H342) is implicated in both protease and esterase activity, suggesting YxiM could be a multifunctional hydrolase. The catalytic motif of the esterase 1BWR aligns particularly well with YxiM.<br />
<br />
We tested YxiM for esterase activity ''in vitro'' in an effort to confirm the ''in silico'' predictions. YxiM showed esterase activity on 4-nitrophenyl butyrate, as absorbance increased during the assay. The Lineweaver-Burk plot of YxiM esterase activity is linear, which is typical of enzymes.<br />
<br />
Thus, on the basis of protein sequence and structural analysis ''in silico'' and functional assays ''in vitro'', we conclude that YxiM is an esterase.<br />
<br />
== Future Directions ==<br />
<br />
To further confirm the activity of YxiM as an esterase, we can perform mutagenesis on the putative catalytic triad by performing PCR on the plasmid DNA with specialized primers. If we mutate the catalytic triad, then we expect that the protein will not be able to perform its function anymore. Through another round of transformation and purification of this mutated DNA, we would expect the protein to show no activity in our esterase assay.<br />
<br />
Our analysis suggests that YxiM could also demonstrate protease activity. To test this, we could perform protease assays as well.<br />
<br />
To further study enzyme kinetics, we could relate absorbance with concentration of protein. We can achieve this by performing a Bradford protein assay to compute the extinction coefficient.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_1&diff=2904791User:Jennifer Taylor/Sandbox 12018-05-25T12:48:11Z<p>Jennifer Taylor: </p>
<hr />
<div>==YxiM from ''Bacillus subtilis''==<br />
<br />
<StructureSection load='2o14' size='340' side='right' caption='Structure of YxiM' scene=''><br />
<br />
== Background ==<br />
<br />
Proteins are an important type of macromolecule in biological systems and can be considered a sequence of subunits known as amino acids. The development of high-throughput genome squencing techniques allowed proteins to be sequenced more quickly than their structures could be solved. In an effort to close this gap, in 2000, the National Institutes of Health launched the 15-year Protein Structure Initiative. Many structures were deposited in the Protein Data Bank, but many of these proteins with solved structures, such as YxiM ([https://www.rcsb.org/structure/2o14 PDB ID: 2O14]), remain functionally uncharacterized. YxiM is transcribed by the ''yxiM'' gene from ''Bacillus subtilis'', a ubiquitous bacterial species that dwells in soil and gastrointestinal tracts. <scene name='78/787191/2o14_rainbow/2'>YxiM</scene> is 375 amino acids in length and its molecular weight is 41.8 kDa. It appears to have two domains: <scene name='78/787191/2o14_alpha/1'>one dominated by α-helices</scene>, and <scene name='78/787191/2o14_beta/1'>one by β-sheets</scene>.<br />
<br />
== ''In silico'' Analysis ==<br />
<br />
A common theme in biology is that form follows function. Thus, we used computer programs to find which proteins were most homologous to YxiM in terms of sequence and structure, with the expectation that YxiM is likely to be functionally similar to those proteins that have similar sequences and structures.<br />
<br />
[[Image:YxiM_green-1J00_blue.png|thumb|left|250px|'''Figure 1'''. YxiM (green) aligned with 1J00 (blue), RMSD = 2.692.]]<br />
<br />
We used BLAST and PFam to find characterized proteins whose sequences aligned best with YxiM. Sequence analysis suggests that YxiM is a GDSL-like lipase, a type of esterase. Esterases are molecules that hydrolyze (decompose) a class of organic molecules known as esters. GDSL-like lipases demonstrate broad substrate specificity due to their flexible structures. BLAST showed that the proteins 1J00, 1IVN, and 1JRL have the highest sequence homology to YxiM. These proteins are multifunctional hydrolases that show both esterase and protease activity.<br />
<br />
Next, we used PyMOL to align the 3D structures of the BLAST hits with that of YxiM. The proteins 1J00, 1IVN, and 1JRL all align well with the α-helix domain of YxiM.<br />
<br />
[[Image:YxiM-1BWR_Catalytic.png|thumb|right|250px|'''Figure 2'''. Active site of 1BWR aligned with the putative active site of YxiM.]]<br />
<br />
The Dali server finds the most similar proteins based on 3D structures, and the top 30 hits for YxiM were are all rhamnogalacturonan acetylesterases, GDSL lipases, LAE5s (hydrolases), or acetyl xylan esterases, which further suggests that YxiM is an esterase.<br />
<br />
Finally, we used ProMOL to perform a structural alignment of active sites of other proteins with YxiM to predict the active site of YxiM. We found that YxiM aligns best with the active site of IBWR, which is an esterase. The <scene name='78/787191/2o14_active_site/2'>putative catalytic triad</scene> of YxiM consists of amino acids S171, D339, and H342.<br />
<br />
Based on these analyses, we predicted that YxiM is an esterase and proceeded to perform ''in vitro'' assays to confirm esterase activity.<br />
<br />
== Plasmid Purification ==<br />
<br />
[[Image:PET21-YxiM_Map.png|thumb|right|250px|'''Figure 3'''. Plasmid map of pET21-''yxiM''.]]<br />
<br />
In order to study the protein YxiM, we ordered a plasmid that contains the gene that transcribes the protein. A plasmid is a type of circular bacterial DNA. By transforming (inserting) this plasmid (pET21-''yxiM'') into the bacteria (DH5α Competent ''E. coli''), we can use the bacteria to create more of the plasmid. Then, we performed a DNA miniprep to purify the plasmid for later use.<br />
<br />
== Bacterial Transformation ==<br />
<br />
While DH5α ''E. coli'' are good for purifying plasmids, BL21(DE3) ''E. coli'' are more efficient for expressing protein. Thus, we transformed the plasmid into BL21(DE3) for the purposes of protein expression. We plated the bacteria on agar with the antibiotic ampicillin. While normal ''E. coli'' will die in the presence of ampicillin, the pET21-''yxiM'' plasmid has a gene that allows bacteria to become ampicillin resistant. Thus, only bacteria that were successfully transformed by the plasmid will survive on the ampicillin plate, allowing us to select for bacteria that have been transformed and thus bacteria that will express the protein YxiM.<br />
<br />
== Protein Expression ==<br />
<br />
After a day, colonies of transformed bacteria were visible on the agar plates. To express YxiM, we inoculated a single colony of bacteria into a liquid culture. In the plasmid, the ''yxiM'' gene is under control of the ''lac'' operon. This means that in the absence of an inducer, the transcription of the ''yxiM'' gene is repressed. Thus, we added IPTG, to activate the operon and drive the transcription of the protein YxiM.<br />
<br />
== Protein Purification ==<br />
<br />
After several hours, the bacteria have produced a relatively large amount of YxiM. To collect the protein, which at this point remained inside the cells, the bacteria were lysed (their cell walls were burst). The resulting mixture consisted of various cellular proteins and debris. The plasmid DNA sequence that coded for YxiM added a "tag" of histidines at the end of the protein. This allows us to separate YxiM proteins from the other types of proteins in the ''E. coli'' cells because when the mixture is passed through a nickel column, the tagged YxiM proteins stick to the column, while the other proteins flow through. Finally, we added elution buffer to the columns, which caused the proteins to detach from the nickel columns, creating a solution of just the YxiM proteins.<br />
<br />
== Esterase Activity Assay ==<br />
<br />
[[Image:YxiM_Lineweaver-Burk_Plot.png|thumb|left|250px|'''Figure 4'''. Lineweaver-Burk plot of YxiM esterase activity.]]<br />
<br />
Now that we had purified protein, we could test the function of YxiM ''in vitro''. Since we believed that YxiM was an ester, we placed it in a buffered solution with 4-nitrophenyl butyrate, a type of ester. Esterases should hydrolyze 4-nitrophenyl butyrate, causing the products butyric acid and 4-nitrophenol to form. Since 4-nitrophenyl is a yellow color, the absorbance of the solution changes as more products are formed. We used colorimeter to measure the absorbance at 430 nm as a proxy for esterase activity. We found that the absorbance increases over time, which suggests that YxiM is indeed an esterase.<br />
<br />
Specifically, we found that the Lineweaver-Burk plot of esterase activity is linear. This is typical of enzymes, as predicted by the Michaelis-Menten model of enzyme kinetics. Technically, we did not construct a true Lineweaver-Burk plot, as we used absorbance as a proxy for molar concentration, but absorbance varies linearly with concentration, as shown by the Beer-Lambert law.<br />
<br />
== Discussion ==<br />
<br />
YxiM is a previously uncharacterized protein whose crystal structure has been solved and deposited in the PDB.<br />
<br />
Protein sequence analysis with BLAST shows that YxiM is likely an esterase. PyMOL shows that the top BLAST hits also align well with the 3D structure of the α-helix domain of YxiM. Almost all the top structural hits in Dali are esterases as well, and ProMOL shows that the active site of YxiM most resembles one of an esterase or protease. The same catalytic triad (S171, D339, H342) is implicated in both protease and esterase activity, suggesting YxiM could be a multifunctional hydrolase. The catalytic motif of the esterase 1BWR aligns particularly well with YxiM.<br />
<br />
We tested YxiM for esterase activity ''in vitro'' in an effort to confirm the ''in silico'' predictions. YxiM showed esterase activity on 4-nitrophenyl butyrate, as absorbance increased during the assay. The Lineweaver-Burk plot of YxiM esterase activity is linear, which is typical of enzymes.<br />
<br />
Thus, on the basis of protein sequence and structural analysis ''in silico'' and functional assays ''in vitro'', we conclude that YxiM is an esterase.<br />
<br />
== Future Directions ==<br />
<br />
To further confirm the activity of YxiM as an esterase, we can perform mutagenesis on the putative catalytic triad by performing PCR on the plasmid DNA with specialized primers. If we mutate the catalytic triad, then we expect that the protein will not be able to perform its function anymore. Through another round of transformation and purification of this mutated DNA, we would expect the protein to show no activity in our esterase assay.<br />
<br />
Our analysis suggests that YxiM could also demonstrate protease activity. To test this, we could perform protease assays as well.<br />
<br />
To further study enzyme kinetics, we could relate absorbance with concentration of protein. We can achieve this by performing a Bradford protein assay to compute the extinction coefficient.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_7&diff=2904790User:Jennifer Taylor/Sandbox 72018-05-25T12:27:20Z<p>Jennifer Taylor: </p>
<hr />
<div>==About 4Q7Q:A Hydrolase==<br />
<StructureSection load='4q7q' size='340' side='both' caption='Caption for this structure' scene=''><br />
This is a default text for your page '''Jennifer Taylor/Sandbox 7'''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br />
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br />
<br />
==What is 4Q7Q?==<br />
In 2000, the protein structure initiative (PSI) began with the goal of finding the three dimensional structure of as many proteins as possible. In 2010, however, funding for the PSI was terminated. Nevertheless, millions of protein structures were found, and many structures were uncharacterized. One of these uncharacterized proteins was <scene name='78/787195/4q7q_assymetric_assembly/1'>4Q7Q</scene>, which is found in <i>Chitinophaga pinesis</i>. Its molecular weight is 87.1 kDa. Through structural and sequential analysis on BLAST, PFam, Dali, and ProMol, 4Q7Q is believed to be a lipase.<br />
==Sequential analysis of 4Q7Q==<br />
<b>BLAST</b>-Sequence homology between 4Q7Q was tested through BLAST; the top hit for 4Q7Q was <scene name='78/787195/4m8k/1'>4M8K</scene>, a GDSL-like lipase. 4Q7Q and 4M8k share a 36% sequence homology, but a low E-value, indicating it is a significant match. <br />
<br />
<b>PFam</b>-Results from PFam sequence mapping also indicated that 4Q7Q shares sequence homology with other GDSL-like lipases. <br />
<br />
<b>Snapgene</b>-Snapgene was able to reveal the molecular mass of 4Q7Q, later used as a reference when its purified form was run on an SDS-page gel. Snapgene also provided information about tags used to insert its gene into the plasmid used for transformation in DH5α and BL21 E. coli cells.<br />
<br />
[[Image:snapgene_map.png|thumb|left|350px|Figure 1: Snapgene map of 4Q7Q plasmid. 4Q7Q sequence information can be found on the top left corner of the image.]] {{clear}}<br />
== Structural analysis of 4Q7Q==<br />
<b>Dali</b>-Top hits from Dali indicated that 4Q7Q shares structural homology with putative lipases.<br />
<br />
<b>ProMol</b>-Through ProMol, a possible catalytic triad for <scene name='78/787195/4q7q_catalytic_triad/1'>4Q7Q</scene> (Ser164, Asp193, His196) was found due to catalytic triad alignment with 1BRW. 4Q7Q and 1BRW catalytic triad alignment yielded a RMS value of 2.049, and a full structural homology with an RMS value of 4.852.<br />
<br />
==Enzymatic Assay of 4Q7Q==<br />
<b>Reasoning</b>-Results obtained in silico strongly suggested that 4Q7Q was most likely to be hydrolase, and specifically a lipase. Thus, a lipase assay was run on 4Q7Q to confirm its enzymatic function. The lipase assay conducted was done through spectrophotometric methods. Purified 4Q7Q was mixed in solution with P-nitrophenyl butyrate. When nitrophenyl butyrate undergoes hydrolysis, its products are p-nitrophenol and butyrate. P-nitrophenol’s color becomes yellow when this reaction happens in solution.Thus, when a lipase catalyzes the hydrolysis of P-nitrophenol butyrate, a color change in solution will occur. A colorimeter was set at 430nm to detect light in the “yellow” range and thereby measure the change in color occuring when 4Q7Q catalyzed the hydrolysis reaction.<br />
<br />
<b>4.2 Results</b><br />
[[Image:4q7q_SDS.png|thumb|left|350px|Figure 2: SDS-PAGE gel of purified 4Q7Q plasmid.]] {{clear}}<br />
<br />
[[Image:pNPB assay.png|thumb|left|350px|Figure 3:pNPB Assay Results for 4Q7Q.]] {{clear}}<br />
<br />
<b>Conclusion</b>-Due to 4Q7Q’s low concentration found on the SDS-page gel, 4Q7Q to pNPB ratio in solution was 10:1. A logarithmic-curve was present during the lipase assay conducted on 4Q7Q, indicating it successfully catalyzed the hydrolysis of a lipid. <br />
==Future directions==<br />
<b>Cloning</b>-To confirm the suspected catalytic triad of 4Q7Q, site directed mutagenesis of 4Q7Qs catalytic triad was performed through PCR. Substitutions of residues were completed as followed: Ser164Ala, Asp193Ala, His196Ala. Following a successful cloning, a p-NPB lipase assay will be performed to see whether the new mutations on 4Q7Q deterred catalysis of the hydrolysis reaction of p-NPB.<br />
<br />
[[Image:4Q7Q_Mutagenesis.png|thumb|left|350px|Figure 3:Mutagenesis of 4Q7Q (Ser164Ala, Asp193Ala, His196Ala).]] {{clear}}<br />
<br />
<b>Enzyme Kinetics of 4Q7Q</b>=Further data of enzyme activity of 4Q7Q using our lipase assay at different concentrations of p-NPB can be performed to create a Lineweaver-Burk plot.<br />
<br />
<b>Optimization of enzymatic activity</b>-Conditions for optimized enzyme activity can be tested by doing pH and temperature iterations of the p-NPB assay.<br />
<br />
<br />
<br />
<br />
</StructureSection><br />
== References and Notes ==<br />
<references/></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=File:4Q7Q_Mutagenesis.png&diff=2904789File:4Q7Q Mutagenesis.png2018-05-25T12:26:26Z<p>Jennifer Taylor: </p>
<hr />
<div>== Licensing ==<br />
{{self|cc-by-3.0}}</div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_7&diff=2904788User:Jennifer Taylor/Sandbox 72018-05-25T12:25:39Z<p>Jennifer Taylor: </p>
<hr />
<div>==About 4Q7Q:A Hydrolase==<br />
<StructureSection load='4q7q' size='340' side='both' caption='Caption for this structure' scene=''><br />
This is a default text for your page '''Jennifer Taylor/Sandbox 7'''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br />
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br />
<br />
==What is 4Q7Q?==<br />
In 2000, the protein structure initiative (PSI) began with the goal of finding the three dimensional structure of as many proteins as possible. In 2010, however, funding for the PSI was terminated. Nevertheless, millions of protein structures were found, and many structures were uncharacterized. One of these uncharacterized proteins was <scene name='78/787195/4q7q_assymetric_assembly/1'>4Q7Q</scene>, which is found in <i>Chitinophaga pinesis</i>. Its molecular weight is 87.1 kDa. Through structural and sequential analysis on BLAST, PFam, Dali, and ProMol, 4Q7Q is believed to be a lipase.<br />
==Sequential analysis of 4Q7Q==<br />
<b>BLAST</b>-Sequence homology between 4Q7Q was tested through BLAST; the top hit for 4Q7Q was <scene name='78/787195/4m8k/1'>4M8K</scene>, a GDSL-like lipase. 4Q7Q and 4M8k share a 36% sequence homology, but a low E-value, indicating it is a significant match. <br />
<br />
<b>PFam</b>-Results from PFam sequence mapping also indicated that 4Q7Q shares sequence homology with other GDSL-like lipases. <br />
<br />
<b>Snapgene</b>-Snapgene was able to reveal the molecular mass of 4Q7Q, later used as a reference when its purified form was run on an SDS-page gel. Snapgene also provided information about tags used to insert its gene into the plasmid used for transformation in DH5α and BL21 E. coli cells.<br />
<br />
[[Image:snapgene_map.png|thumb|left|350px|Figure 1: Snapgene map of 4Q7Q plasmid. 4Q7Q sequence information can be found on the top left corner of the image.]] {{clear}}<br />
== Structural analysis of 4Q7Q==<br />
<b>Dali</b>-Top hits from Dali indicated that 4Q7Q shares structural homology with putative lipases.<br />
<br />
<b>ProMol</b>-Through ProMol, a possible catalytic triad for <scene name='78/787195/4q7q_catalytic_triad/1'>4Q7Q</scene> (Ser164, Asp193, His196) was found due to catalytic triad alignment with 1BRW. 4Q7Q and 1BRW catalytic triad alignment yielded a RMS value of 2.049, and a full structural homology with an RMS value of 4.852.<br />
<br />
==Enzymatic Assay of 4Q7Q==<br />
<b>Reasoning</b>-Results obtained in silico strongly suggested that 4Q7Q was most likely to be hydrolase, and specifically a lipase. Thus, a lipase assay was run on 4Q7Q to confirm its enzymatic function. The lipase assay conducted was done through spectrophotometric methods. Purified 4Q7Q was mixed in solution with P-nitrophenyl butyrate. When nitrophenyl butyrate undergoes hydrolysis, its products are p-nitrophenol and butyrate. P-nitrophenol’s color becomes yellow when this reaction happens in solution.Thus, when a lipase catalyzes the hydrolysis of P-nitrophenol butyrate, a color change in solution will occur. A colorimeter was set at 430nm to detect light in the “yellow” range and thereby measure the change in color occuring when 4Q7Q catalyzed the hydrolysis reaction.<br />
<br />
<b>4.2 Results</b><br />
[[Image:4q7q_SDS.png|thumb|left|350px|Figure 2: SDS-PAGE gel of purified 4Q7Q plasmid.]] {{clear}}<br />
<br />
[[Image:pNPB assay.png|thumb|left|350px|Figure 3:pNPB Assay Results for 4Q7Q.]] {{clear}}<br />
<br />
<b>Conclusion</b>-Due to 4Q7Q’s low concentration found on the SDS-page gel, 4Q7Q to pNPB ratio in solution was 10:1. A logarithmic-curve was present during the lipase assay conducted on 4Q7Q, indicating it successfully catalyzed the hydrolysis of a lipid. <br />
==Future directions==<br />
<b>Cloning</b>-To confirm the suspected catalytic triad of 4Q7Q, site directed mutagenesis of 4Q7Qs catalytic triad was performed through PCR. Substitutions of residues were completed as followed: Ser164Ala, Asp193Ala, His196Ala. Following a successful cloning, a p-NPB lipase assay will be performed to see whether the new mutations on 4Q7Q deterred catalysis of the hydrolysis reaction of p-NPB.<br />
<br />
<b>Enzyme Kinetics of 4Q7Q</b>=Further data of enzyme activity of 4Q7Q using our lipase assay at different concentrations of p-NPB can be performed to create a Lineweaver-Burk plot.<br />
<br />
<b>Optimization of enzymatic activity</b>-Conditions for optimized enzyme activity can be tested by doing pH and temperature iterations of the p-NPB assay.<br />
<br />
<br />
<br />
<br />
</StructureSection><br />
== References and Notes ==<br />
<references/></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=File:PNPB_assay.png&diff=2904787File:PNPB assay.png2018-05-25T12:22:10Z<p>Jennifer Taylor: </p>
<hr />
<div>== Licensing ==<br />
{{self|cc-by-3.0}}</div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_7&diff=2904786User:Jennifer Taylor/Sandbox 72018-05-25T12:18:25Z<p>Jennifer Taylor: </p>
<hr />
<div>==About 4Q7Q:A Hydrolase==<br />
<StructureSection load='4q7q' size='340' side='both' caption='Caption for this structure' scene=''><br />
This is a default text for your page '''Jennifer Taylor/Sandbox 7'''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br />
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br />
<br />
==What is 4Q7Q?==<br />
In 2000, the protein structure initiative (PSI) began with the goal of finding the three dimensional structure of as many proteins as possible. In 2010, however, funding for the PSI was terminated. Nevertheless, millions of protein structures were found, and many structures were uncharacterized. One of these uncharacterized proteins was <scene name='78/787195/4q7q_assymetric_assembly/1'>4Q7Q</scene>, which is found in <i>Chitinophaga pinesis</i>. Its molecular weight is 87.1 kDa. Through structural and sequential analysis on BLAST, PFam, Dali, and ProMol, 4Q7Q is believed to be a lipase.<br />
==Sequential analysis of 4Q7Q==<br />
<b>BLAST</b>-Sequence homology between 4Q7Q was tested through BLAST; the top hit for 4Q7Q was <scene name='78/787195/4m8k/1'>4M8K</scene>, a GDSL-like lipase. 4Q7Q and 4M8k share a 36% sequence homology, but a low E-value, indicating it is a significant match. <br />
<br />
<b>PFam</b>-Results from PFam sequence mapping also indicated that 4Q7Q shares sequence homology with other GDSL-like lipases. <br />
<br />
<b>Snapgene</b>-Snapgene was able to reveal the molecular mass of 4Q7Q, later used as a reference when its purified form was run on an SDS-page gel. Snapgene also provided information about tags used to insert its gene into the plasmid used for transformation in DH5α and BL21 E. coli cells.<br />
<br />
[[Image:snapgene_map.png|thumb|left|350px|Figure 1: Snapgene map of 4Q7Q plasmid. 4Q7Q sequence information can be found on the top left corner of the image.]] {{clear}}<br />
== Structural analysis of 4Q7Q==<br />
<b>Dali</b>-Top hits from Dali indicated that 4Q7Q shares structural homology with putative lipases.<br />
<br />
<b>ProMol</b>-Through ProMol, a possible catalytic triad for <scene name='78/787195/4q7q_catalytic_triad/1'>4Q7Q</scene> (Ser164, Asp193, His196) was found due to catalytic triad alignment with 1BRW. 4Q7Q and 1BRW catalytic triad alignment yielded a RMS value of 2.049, and a full structural homology with an RMS value of 4.852.<br />
<br />
==Enzymatic Assay of 4Q7Q==<br />
<b>Reasoning</b>-Results obtained in silico strongly suggested that 4Q7Q was most likely to be hydrolase, and specifically a lipase. Thus, a lipase assay was run on 4Q7Q to confirm its enzymatic function. The lipase assay conducted was done through spectrophotometric methods. Purified 4Q7Q was mixed in solution with P-nitrophenyl butyrate. When nitrophenyl butyrate undergoes hydrolysis, its products are p-nitrophenol and butyrate. P-nitrophenol’s color becomes yellow when this reaction happens in solution.Thus, when a lipase catalyzes the hydrolysis of P-nitrophenol butyrate, a color change in solution will occur. A colorimeter was set at 430nm to detect light in the “yellow” range and thereby measure the change in color occuring when 4Q7Q catalyzed the hydrolysis reaction.<br />
<br />
<b>4.2 Results</b><br />
<br />
<b>Conclusion</b>-Due to 4Q7Q’s low concentration found on the SDS-page gel, 4Q7Q to pNPB ratio in solution was 10:1. A logarithmic-curve was present during the lipase assay conducted on 4Q7Q, indicating it successfully catalyzed the hydrolysis of a lipid. <br />
==Future directions==<br />
<b>Cloning</b>-To confirm the suspected catalytic triad of 4Q7Q, site directed mutagenesis of 4Q7Qs catalytic triad was performed through PCR. Substitutions of residues were completed as followed: Ser164Ala, Asp193Ala, His196Ala. Following a successful cloning, a p-NPB lipase assay will be performed to see whether the new mutations on 4Q7Q deterred catalysis of the hydrolysis reaction of p-NPB.<br />
<br />
<b>Enzyme Kinetics of 4Q7Q</b>=Further data of enzyme activity of 4Q7Q using our lipase assay at different concentrations of p-NPB can be performed to create a Lineweaver-Burk plot.<br />
<br />
<b>Optimization of enzymatic activity</b>-Conditions for optimized enzyme activity can be tested by doing pH and temperature iterations of the p-NPB assay.<br />
<br />
<br />
<br />
<br />
</StructureSection><br />
== References and Notes ==<br />
<references/></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=File:4q7q_SDS.png&diff=2904785File:4q7q SDS.png2018-05-25T12:00:45Z<p>Jennifer Taylor: </p>
<hr />
<div></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_7&diff=2904784User:Jennifer Taylor/Sandbox 72018-05-25T11:56:30Z<p>Jennifer Taylor: </p>
<hr />
<div>==About 4Q7Q:A Hydrolase==<br />
<StructureSection load='4q7q' size='340' side='both' caption='Caption for this structure' scene=''><br />
This is a default text for your page '''Jennifer Taylor/Sandbox 7'''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br />
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br />
<br />
==What is 4Q7Q?==<br />
In 2000, the protein structure initiative (PSI) began with the goal of finding the three dimensional structure of as many proteins as possible. In 2010, however, funding for the PSI was terminated. Nevertheless, millions of protein structures were found, and many structures were uncharacterized. One of these uncharacterized proteins was <scene name='78/787195/4q7q_assymetric_assembly/1'>4Q7Q</scene>, which is found in <i>Chitinophaga pinesis</i>. Its molecular weight is 87.1 kDa. Through structural and sequential analysis on BLAST, PFam, Dali, and ProMol, 4Q7Q is believed to be a lipase.<br />
==Sequential analysis of 4Q7Q==<br />
<b>BLAST</b>-Sequence homology between 4Q7Q was tested through BLAST; the top hit for 4Q7Q was <scene name='78/787195/4m8k/1'>4M8K</scene>, a GDSL-like lipase. 4Q7Q and 4M8k share a 36% sequence homology, but a low E-value, indicating it is a significant match. <br />
<br />
<b>PFam</b>-Results from PFam sequence mapping also indicated that 4Q7Q shares sequence homology with other GDSL-like lipases. <br />
<br />
<b>Snapgene</b>-Snapgene was able to reveal the molecular mass of 4Q7Q, later used as a reference when its purified form was run on an SDS-page gel. Snapgene also provided information about tags used to insert its gene into the plasmid used for transformation in DH5α and BL21 E. coli cells.<br />
<br />
[[Image:snapgene_map.png|thumb|left|350px|Figure 1: Snapgene map of 4Q7Q plasmid. 4Q7Q sequence information can be found on the top left corner of the image.]] {{clear}}<br />
== Structural analysis of 4Q7Q==<br />
<b>Dali</b>-Top hits from Dali indicated that 4Q7Q shares structural homology with putative lipases.<br />
<br />
<b>ProMol</b>-Through ProMol, a possible catalytic triad for 4Q7Q (Ser164, Asp193, His196) was found due to catalytic triad alignment with 1BRW. 4Q7Q and 1BRW catalytic triad alignment yielded a RMS value of 2.049, and a full structural homology with an RMS value of 4.852.<br />
<br />
==Enzymatic Assay of 4Q7Q==<br />
<b>Reasoning</b>-Results obtained in silico strongly suggested that 4Q7Q was most likely to be hydrolase, and specifically a lipase. Thus, a lipase assay was run on 4Q7Q to confirm its enzymatic function. The lipase assay conducted was done through spectrophotometric methods. Purified 4Q7Q was mixed in solution with P-nitrophenyl butyrate. When nitrophenyl butyrate undergoes hydrolysis, its products are p-nitrophenol and butyrate. P-nitrophenol’s color becomes yellow when this reaction happens in solution.Thus, when a lipase catalyzes the hydrolysis of P-nitrophenol butyrate, a color change in solution will occur. A colorimeter was set at 430nm to detect light in the “yellow” range and thereby measure the change in color occuring when 4Q7Q catalyzed the hydrolysis reaction.<br />
<br />
<b>4.2 Results</b><br />
<br />
<b>Conclusion</b>-Due to 4Q7Q’s low concentration found on the SDS-page gel, 4Q7Q to pNPB ratio in solution was 10:1. A logarithmic-curve was present during the lipase assay conducted on 4Q7Q, indicating it successfully catalyzed the hydrolysis of a lipid. <br />
==Future directions==<br />
<b>Cloning</b>-To confirm the suspected catalytic triad of 4Q7Q, site directed mutagenesis of 4Q7Qs catalytic triad was performed through PCR. Substitutions of residues were completed as followed: Ser164Ala, Asp193Ala, His196Ala. Following a successful cloning, a p-NPB lipase assay will be performed to see whether the new mutations on 4Q7Q deterred catalysis of the hydrolysis reaction of p-NPB.<br />
<br />
<b>Enzyme Kinetics of 4Q7Q</b>=Further data of enzyme activity of 4Q7Q using our lipase assay at different concentrations of p-NPB can be performed to create a Lineweaver-Burk plot.<br />
<br />
<b>Optimization of enzymatic activity</b>-Conditions for optimized enzyme activity can be tested by doing pH and temperature iterations of the p-NPB assay.<br />
<br />
<br />
<br />
<br />
</StructureSection><br />
== References and Notes ==<br />
<references/></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_7&diff=2904783User:Jennifer Taylor/Sandbox 72018-05-25T11:53:08Z<p>Jennifer Taylor: </p>
<hr />
<div>==About 4Q7Q:A Hydrolase==<br />
Here is a polar hydrophobic residue image of my protein, <scene name='78/787195/4q7q_polar_and_hydrophobic/1'>4Q7Q</scene>.<br />
<StructureSection load='4q7q' size='340' side='both' caption='Caption for this structure' scene=''><br />
This is a default text for your page '''Jennifer Taylor/Sandbox 7'''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br />
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br />
<br />
==What is 4Q7Q?==<br />
In 2000, the protein structure initiative (PSI) began with the goal of finding the three dimensional structure of as many proteins as possible. In 2010, however, funding for the PSI was terminated. Nevertheless, millions of protein structures were found, and many structures were uncharacterized. One of these uncharacterized proteins was <scene name='78/787195/4q7q_assymetric_assembly/1'>4Q7Q</scene>, which is found in <i>Chitinophaga pinesis</i>. Its molecular weight is 87.1 kDa. Through structural and sequential analysis on BLAST, PFam, Dali, and ProMol, 4Q7Q is believed to be a lipase.<br />
==Sequential analysis of 4Q7Q==<br />
<b>BLAST</b>-Sequence homology between 4Q7Q was tested through BLAST; the top hit for 4Q7Q was 4M8K, a GDSL-like lipase. 4Q7Q and 4M8k share a 36% sequence homology, but a low E-value, indicating it is a significant match. <br />
<br />
<b>PFam</b>-Results from PFam sequence mapping also indicated that 4Q7Q shares sequence homology with other GDSL-like lipases. <br />
<br />
<b>Snapgene</b>-Snapgene was able to reveal the molecular mass of 4Q7Q, later used as a reference when its purified form was run on an SDS-page gel. Snapgene also provided information about tags used to insert its gene into the plasmid used for transformation in DH5α and BL21 E. coli cells.<br />
<br />
[[Image:snapgene_map.png|thumb|left|350px|Figure 1: Snapgene map of 4Q7Q plasmid. 4Q7Q sequence information can be found on the top left corner of the image.]] {{clear}}<br />
== Structural analysis of 4Q7Q==<br />
<b>Dali</b>-Top hits from Dali indicated that 4Q7Q shares structural homology with putative lipases.<br />
<br />
<b>ProMol</b>-Through ProMol, a possible catalytic triad for 4Q7Q (Ser164, Asp193, His196) was found due to catalytic triad alignment with 1BRW. 4Q7Q and 1BRW catalytic triad alignment yielded a RMS value of 2.049, and a full structural homology with an RMS value of 4.852.<br />
<br />
==Enzymatic Assay of 4Q7Q==<br />
<b>Reasoning</b>-Results obtained in silico strongly suggested that 4Q7Q was most likely to be hydrolase, and specifically a lipase. Thus, a lipase assay was run on 4Q7Q to confirm its enzymatic function. The lipase assay conducted was done through spectrophotometric methods. Purified 4Q7Q was mixed in solution with P-nitrophenyl butyrate. When nitrophenyl butyrate undergoes hydrolysis, its products are p-nitrophenol and butyrate. P-nitrophenol’s color becomes yellow when this reaction happens in solution.Thus, when a lipase catalyzes the hydrolysis of P-nitrophenol butyrate, a color change in solution will occur. A colorimeter was set at 430nm to detect light in the “yellow” range and thereby measure the change in color occuring when 4Q7Q catalyzed the hydrolysis reaction.<br />
<br />
<b>4.2 Results</b><br />
<br />
<b>Conclusion</b>-Due to 4Q7Q’s low concentration found on the SDS-page gel, 4Q7Q to pNPB ratio in solution was 10:1. A logarithmic-curve was present during the lipase assay conducted on 4Q7Q, indicating it successfully catalyzed the hydrolysis of a lipid. <br />
==Future directions==<br />
<b>Cloning</b>-To confirm the suspected catalytic triad of 4Q7Q, site directed mutagenesis of 4Q7Qs catalytic triad was performed through PCR. Substitutions of residues were completed as followed: Ser164Ala, Asp193Ala, His196Ala. Following a successful cloning, a p-NPB lipase assay will be performed to see whether the new mutations on 4Q7Q deterred catalysis of the hydrolysis reaction of p-NPB.<br />
<br />
<b>Enzyme Kinetics of 4Q7Q</b>=Further data of enzyme activity of 4Q7Q using our lipase assay at different concentrations of p-NPB can be performed to create a Lineweaver-Burk plot.<br />
<br />
<b>Optimization of enzymatic activity</b>-Conditions for optimized enzyme activity can be tested by doing pH and temperature iterations of the p-NPB assay.<br />
<br />
<br />
<br />
<br />
</StructureSection><br />
== References and Notes ==<br />
<references/></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_7&diff=2904782User:Jennifer Taylor/Sandbox 72018-05-25T11:45:53Z<p>Jennifer Taylor: </p>
<hr />
<div>==About 4Q7Q:A Hydrolase==<br />
Here is a polar hydrophobic residue image of my protein, <scene name='78/787195/4q7q_polar_and_hydrophobic/1'>4Q7Q</scene>.<br />
<StructureSection load='4q7q' size='340' side='both' caption='Caption for this structure' scene=''><br />
This is a default text for your page '''Jennifer Taylor/Sandbox 7'''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br />
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br />
<br />
==What is 4Q7Q?==<br />
In 2000, the protein structure initiative (PSI) began with the goal of finding the three dimensional structure of as many proteins as possible. In 2010, however, funding for the PSI was terminated. Nevertheless, millions of protein structures were found, and many structures were uncharacterized. One of these uncharacterized proteins found in Chitinophaga pinesis. Its molecular weight is 87.1 kDa. Through structural and sequential analysis on BLAST, PFam, Dali, and ProMol, 4Q7Q is believed to be a lipase.<br />
==Sequential analysis of 4Q7Q==<br />
<b>BLAST</b>-Sequence homology between 4Q7Q was tested through BLAST; the top hit for 4Q7Q was 4M8K, a GDSL-like lipase. 4Q7Q and 4M8k share a 36% sequence homology, but a low E-value, indicating it is a significant match. <br />
<br />
<b>PFam</b>-Results from PFam sequence mapping also indicated that 4Q7Q shares sequence homology with other GDSL-like lipases. <br />
<br />
<b>Snapgene</b>-Snapgene was able to reveal the molecular mass of 4Q7Q, later used as a reference when its purified form was run on an SDS-page gel. Snapgene also provided information about tags used to insert its gene into the plasmid used for transformation in DH5α and BL21 E. coli cells.<br />
<br />
[[Image:snapgene_map.png|thumb|left|350px|Figure 1: Snapgene map of 4Q7Q plasmid. 4Q7Q sequence information can be found on the top left corner of the image.]] {{clear}}<br />
== Structural analysis of 4Q7Q==<br />
<b>Dali</b>-Top hits from Dali indicated that 4Q7Q shares structural homology with putative lipases.<br />
<br />
<b>ProMol</b>-Through ProMol, a possible catalytic triad for 4Q7Q (Ser164, Asp193, His196) was found due to catalytic triad alignment with 1BRW. 4Q7Q and 1BRW catalytic triad alignment yielded a RMS value of 2.049, and a full structural homology with an RMS value of 4.852.<br />
<br />
==Enzymatic Assay of 4Q7Q==<br />
<b>Reasoning</b>-Results obtained in silico strongly suggested that 4Q7Q was most likely to be hydrolase, and specifically a lipase. Thus, a lipase assay was run on 4Q7Q to confirm its enzymatic function. The lipase assay conducted was done through spectrophotometric methods. Purified 4Q7Q was mixed in solution with P-nitrophenyl butyrate. When nitrophenyl butyrate undergoes hydrolysis, its products are p-nitrophenol and butyrate. P-nitrophenol’s color becomes yellow when this reaction happens in solution.Thus, when a lipase catalyzes the hydrolysis of P-nitrophenol butyrate, a color change in solution will occur. A colorimeter was set at 430nm to detect light in the “yellow” range and thereby measure the change in color occuring when 4Q7Q catalyzed the hydrolysis reaction.<br />
<br />
<b>4.2 Results</b><br />
<br />
<b>Conclusion</b>-Due to 4Q7Q’s low concentration found on the SDS-page gel, 4Q7Q to pNPB ratio in solution was 10:1. A logarithmic-curve was present during the lipase assay conducted on 4Q7Q, indicating it successfully catalyzed the hydrolysis of a lipid. <br />
==Future directions==<br />
<b>Cloning</b>-To confirm the suspected catalytic triad of 4Q7Q, site directed mutagenesis of 4Q7Qs catalytic triad was performed through PCR. Substitutions of residues were completed as followed: Ser164Ala, Asp193Ala, His196Ala. Following a successful cloning, a p-NPB lipase assay will be performed to see whether the new mutations on 4Q7Q deterred catalysis of the hydrolysis reaction of p-NPB.<br />
<br />
<b>Enzyme Kinetics of 4Q7Q</b>=Further data of enzyme activity of 4Q7Q using our lipase assay at different concentrations of p-NPB can be performed to create a Lineweaver-Burk plot.<br />
<br />
<b>Optimization of enzymatic activity</b>-Conditions for optimized enzyme activity can be tested by doing pH and temperature iterations of the p-NPB assay.<br />
<br />
<br />
<br />
<br />
</StructureSection><br />
== References and Notes ==<br />
<references/></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_7&diff=2904781User:Jennifer Taylor/Sandbox 72018-05-25T11:45:22Z<p>Jennifer Taylor: </p>
<hr />
<div>==About 4Q7Q:A Hydrolase==<br />
Here is a polar hydrophobic residue image of my protein, <scene name='78/787195/4q7q_polar_and_hydrophobic/1'>4Q7Q</scene>.<br />
<StructureSection load='4q7q' size='340' side='both' caption='Caption for this structure' scene=''><br />
This is a default text for your page '''Jennifer Taylor/Sandbox 7'''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br />
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br />
<br />
==What is 4Q7Q?==<br />
In 2000, the protein structure initiative (PSI) began with the goal of finding the three dimensional structure of as many proteins as possible. In 2010, however, funding for the PSI was terminated. Nevertheless, millions of protein structures were found, and many structures were uncharacterized. One of these uncharacterized proteins found in Chitinophaga pinesis. Its molecular weight is 87.1 kDa. Through structural and sequential analysis on BLAST, PFam, Dali, and ProMol, 4Q7Q is believed to be a lipase.<br />
==Sequential analysis of 4Q7Q==<br />
<b>BLAST</b>-Sequence homology between 4Q7Q was tested through BLAST; the top hit for 4Q7Q was 4M8K, a GDSL-like lipase. 4Q7Q and 4M8k share a 36% sequence homology, but a low E-value, indicating it is a significant match. <br />
<br />
<b>PFam</b>-Results from PFam sequence mapping also indicated that 4Q7Q shares sequence homology with other GDSL-like lipases. <br />
<br />
<b>Snapgene</b>-Snapgene was able to reveal the molecular mass of 4Q7Q, later used as a reference when its purified form was run on an SDS-page gel. Snapgene also provided information about tags used to insert its gene into the plasmid used for transformation in DH5α and BL21 E. coli cells.<br />
{{Clear}}<br />
[[Image:snapgene_map.png|thumb|left|250px|Figure 1: Snapgene map of 4Q7Q plasmid. 4Q7Q sequence information can be found on the top left corner of the image.]] {{clear}}<br />
== Structural analysis of 4Q7Q==<br />
<b>Dali</b>-Top hits from Dali indicated that 4Q7Q shares structural homology with putative lipases.<br />
<br />
<b>ProMol</b>-Through ProMol, a possible catalytic triad for 4Q7Q (Ser164, Asp193, His196) was found due to catalytic triad alignment with 1BRW. 4Q7Q and 1BRW catalytic triad alignment yielded a RMS value of 2.049, and a full structural homology with an RMS value of 4.852.<br />
<br />
==Enzymatic Assay of 4Q7Q==<br />
<b>Reasoning</b>-Results obtained in silico strongly suggested that 4Q7Q was most likely to be hydrolase, and specifically a lipase. Thus, a lipase assay was run on 4Q7Q to confirm its enzymatic function. The lipase assay conducted was done through spectrophotometric methods. Purified 4Q7Q was mixed in solution with P-nitrophenyl butyrate. When nitrophenyl butyrate undergoes hydrolysis, its products are p-nitrophenol and butyrate. P-nitrophenol’s color becomes yellow when this reaction happens in solution.Thus, when a lipase catalyzes the hydrolysis of P-nitrophenol butyrate, a color change in solution will occur. A colorimeter was set at 430nm to detect light in the “yellow” range and thereby measure the change in color occuring when 4Q7Q catalyzed the hydrolysis reaction.<br />
<br />
<b>4.2 Results</b><br />
<br />
<b>Conclusion</b>-Due to 4Q7Q’s low concentration found on the SDS-page gel, 4Q7Q to pNPB ratio in solution was 10:1. A logarithmic-curve was present during the lipase assay conducted on 4Q7Q, indicating it successfully catalyzed the hydrolysis of a lipid. <br />
==Future directions==<br />
<b>Cloning</b>-To confirm the suspected catalytic triad of 4Q7Q, site directed mutagenesis of 4Q7Qs catalytic triad was performed through PCR. Substitutions of residues were completed as followed: Ser164Ala, Asp193Ala, His196Ala. Following a successful cloning, a p-NPB lipase assay will be performed to see whether the new mutations on 4Q7Q deterred catalysis of the hydrolysis reaction of p-NPB.<br />
<br />
<b>Enzyme Kinetics of 4Q7Q</b>=Further data of enzyme activity of 4Q7Q using our lipase assay at different concentrations of p-NPB can be performed to create a Lineweaver-Burk plot.<br />
<br />
<b>Optimization of enzymatic activity</b>-Conditions for optimized enzyme activity can be tested by doing pH and temperature iterations of the p-NPB assay.<br />
<br />
<br />
<br />
<br />
</StructureSection><br />
== References and Notes ==<br />
<references/></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_7&diff=2904780User:Jennifer Taylor/Sandbox 72018-05-25T11:44:34Z<p>Jennifer Taylor: </p>
<hr />
<div>==About 4Q7Q:A Hydrolase==<br />
Here is a polar hydrophobic residue image of my protein, <scene name='78/787195/4q7q_polar_and_hydrophobic/1'>4Q7Q</scene>.<br />
<StructureSection load='4q7q' size='340' side='both' caption='Caption for this structure' scene=''><br />
This is a default text for your page '''Jennifer Taylor/Sandbox 7'''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br />
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br />
<br />
==What is 4Q7Q?==<br />
In 2000, the protein structure initiative (PSI) began with the goal of finding the three dimensional structure of as many proteins as possible. In 2010, however, funding for the PSI was terminated. Nevertheless, millions of protein structures were found, and many structures were uncharacterized. One of these uncharacterized proteins found in Chitinophaga pinesis. Its molecular weight is 87.1 kDa. Through structural and sequential analysis on BLAST, PFam, Dali, and ProMol, 4Q7Q is believed to be a lipase.<br />
==Sequential analysis of 4Q7Q==<br />
<b>BLAST</b>-Sequence homology between 4Q7Q was tested through BLAST; the top hit for 4Q7Q was 4M8K, a GDSL-like lipase. 4Q7Q and 4M8k share a 36% sequence homology, but a low E-value, indicating it is a significant match. <br />
<br />
<b>PFam</b>-Results from PFam sequence mapping also indicated that 4Q7Q shares sequence homology with other GDSL-like lipases. <br />
<br />
<b>Snapgene</b>-Snapgene was able to reveal the molecular mass of 4Q7Q, later used as a reference when its purified form was run on an SDS-page gel. Snapgene also provided information about tags used to insert its gene into the plasmid used for transformation in DH5α and BL21 E. coli cells.<br />
{{Clear}}<br />
[[Image:snapgene_map.png|thumb|left|250px|Figure 1: Snapgene map of 4Q7Q plasmid. 4Q7Q sequence information can be found on the top left corner of the image.]]<br />
== Structural analysis of 4Q7Q==<br />
<b>Dali</b>-Top hits from Dali indicated that 4Q7Q shares structural homology with putative lipases.<br />
<br />
<b>ProMol</b>-Through ProMol, a possible catalytic triad for 4Q7Q (Ser164, Asp193, His196) was found due to catalytic triad alignment with 1BRW. 4Q7Q and 1BRW catalytic triad alignment yielded a RMS value of 2.049, and a full structural homology with an RMS value of 4.852.<br />
<br />
==Enzymatic Assay of 4Q7Q==<br />
<b>Reasoning</b>-Results obtained in silico strongly suggested that 4Q7Q was most likely to be hydrolase, and specifically a lipase. Thus, a lipase assay was run on 4Q7Q to confirm its enzymatic function. The lipase assay conducted was done through spectrophotometric methods. Purified 4Q7Q was mixed in solution with P-nitrophenyl butyrate. When nitrophenyl butyrate undergoes hydrolysis, its products are p-nitrophenol and butyrate. P-nitrophenol’s color becomes yellow when this reaction happens in solution.Thus, when a lipase catalyzes the hydrolysis of P-nitrophenol butyrate, a color change in solution will occur. A colorimeter was set at 430nm to detect light in the “yellow” range and thereby measure the change in color occuring when 4Q7Q catalyzed the hydrolysis reaction.<br />
<br />
<b>4.2 Results</b><br />
<br />
<b>Conclusion</b>-Due to 4Q7Q’s low concentration found on the SDS-page gel, 4Q7Q to pNPB ratio in solution was 10:1. A logarithmic-curve was present during the lipase assay conducted on 4Q7Q, indicating it successfully catalyzed the hydrolysis of a lipid. <br />
==Future directions==<br />
<b>Cloning</b>-To confirm the suspected catalytic triad of 4Q7Q, site directed mutagenesis of 4Q7Qs catalytic triad was performed through PCR. Substitutions of residues were completed as followed: Ser164Ala, Asp193Ala, His196Ala. Following a successful cloning, a p-NPB lipase assay will be performed to see whether the new mutations on 4Q7Q deterred catalysis of the hydrolysis reaction of p-NPB.<br />
<br />
<b>Enzyme Kinetics of 4Q7Q</b>=Further data of enzyme activity of 4Q7Q using our lipase assay at different concentrations of p-NPB can be performed to create a Lineweaver-Burk plot.<br />
<br />
<b>Optimization of enzymatic activity</b>-Conditions for optimized enzyme activity can be tested by doing pH and temperature iterations of the p-NPB assay.<br />
<br />
<br />
<br />
<br />
</StructureSection><br />
== References and Notes ==<br />
<references/></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=File:Snapgene_map.png&diff=2904778File:Snapgene map.png2018-05-25T11:38:44Z<p>Jennifer Taylor: </p>
<hr />
<div>== Licensing ==<br />
{{self|cc-by-3.0}}</div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_7&diff=2904777User:Jennifer Taylor/Sandbox 72018-05-25T11:35:28Z<p>Jennifer Taylor: </p>
<hr />
<div>==About 4Q7Q:A Hydrolase==<br />
Here is a polar hydrophobic residue image of my protein, <scene name='78/787195/4q7q_polar_and_hydrophobic/1'>4Q7Q</scene>.<br />
<StructureSection load='4q7q' size='340' side='both' caption='Caption for this structure' scene=''><br />
This is a default text for your page '''Jennifer Taylor/Sandbox 7'''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br />
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br />
<br />
==What is 4Q7Q?==<br />
In 2000, the protein structure initiative (PSI) began with the goal of finding the three dimensional structure of as many proteins as possible. In 2010, however, funding for the PSI was terminated. Nevertheless, millions of protein structures were found, and many structures were uncharacterized. One of these uncharacterized proteins found in Chitinophaga pinesis. Its molecular weight is 87.1 kDa. Through structural and sequential analysis on BLAST, PFam, Dali, and ProMol, 4Q7Q is believed to be a lipase.<br />
==Sequential analysis of 4Q7Q==<br />
<b>BLAST</b>-Sequence homology between 4Q7Q was tested through BLAST; the top hit for 4Q7Q was 4M8K, a GDSL-like lipase. 4Q7Q and 4M8k share a 36% sequence homology, but a low E-value, indicating it is a significant match. <br />
<br />
<b>PFam</b>-Results from PFam sequence mapping also indicated that 4Q7Q shares sequence homology with other GDSL-like lipases. <br />
<br />
<b>Snapgene</b>-Snapgene was able to reveal the molecular mass of 4Q7Q, later used as a reference when its purified form was run on an SDS-page gel. Snapgene also provided information about tags used to insert its gene into the plasmid used for transformation in DH5α and BL21 E. coli cells.<br />
== Structural analysis of 4Q7Q==<br />
<b>Dali</b>-Top hits from Dali indicated that 4Q7Q shares structural homology with putative lipases.<br />
<br />
<b>ProMol</b>-Through ProMol, a possible catalytic triad for 4Q7Q (Ser164, Asp193, His196) was found due to catalytic triad alignment with 1BRW. 4Q7Q and 1BRW catalytic triad alignment yielded a RMS value of 2.049, and a full structural homology with an RMS value of 4.852.<br />
<br />
==Enzymatic Assay of 4Q7Q==<br />
<b>Reasoning</b>-Results obtained in silico strongly suggested that 4Q7Q was most likely to be hydrolase, and specifically a lipase. Thus, a lipase assay was run on 4Q7Q to confirm its enzymatic function. The lipase assay conducted was done through spectrophotometric methods. Purified 4Q7Q was mixed in solution with P-nitrophenyl butyrate. When nitrophenyl butyrate undergoes hydrolysis, its products are p-nitrophenol and butyrate. P-nitrophenol’s color becomes yellow when this reaction happens in solution.Thus, when a lipase catalyzes the hydrolysis of P-nitrophenol butyrate, a color change in solution will occur. A colorimeter was set at 430nm to detect light in the “yellow” range and thereby measure the change in color occuring when 4Q7Q catalyzed the hydrolysis reaction.<br />
<br />
<b>4.2 Results</b><br />
<br />
<b>Conclusion</b>-Due to 4Q7Q’s low concentration found on the SDS-page gel, 4Q7Q to pNPB ratio in solution was 10:1. A logarithmic-curve was present during the lipase assay conducted on 4Q7Q, indicating it successfully catalyzed the hydrolysis of a lipid. <br />
==Future directions==<br />
<b>Cloning</b>-To confirm the suspected catalytic triad of 4Q7Q, site directed mutagenesis of 4Q7Qs catalytic triad was performed through PCR. Substitutions of residues were completed as followed: Ser164Ala, Asp193Ala, His196Ala. Following a successful cloning, a p-NPB lipase assay will be performed to see whether the new mutations on 4Q7Q deterred catalysis of the hydrolysis reaction of p-NPB.<br />
<br />
<b>Enzyme Kinetics of 4Q7Q</b>=Further data of enzyme activity of 4Q7Q using our lipase assay at different concentrations of p-NPB can be performed to create a Lineweaver-Burk plot.<br />
<br />
<b>Optimization of enzymatic activity</b>-Conditions for optimized enzyme activity can be tested by doing pH and temperature iterations of the p-NPB assay.<br />
<br />
<br />
<br />
<br />
</StructureSection><br />
== References and Notes ==<br />
<references/></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_7&diff=2904776User:Jennifer Taylor/Sandbox 72018-05-25T11:29:07Z<p>Jennifer Taylor: </p>
<hr />
<div>==About 4Q7Q:A Hydrolase==<br />
Here is a polar hydrophobic residue image of my protein, <scene name='78/787195/4q7q_polar_and_hydrophobic/1'>4Q7Q</scene>.<br />
<StructureSection load='4q7q' size='340' side='both' caption='Caption for this structure' scene=''><br />
This is a default text for your page '''Jennifer Taylor/Sandbox 7'''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br />
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br />
<br />
==What is 4Q7Q?==<br />
In 2000, the protein structure initiative (PSI) began with the goal of finding the three dimensional structure of as many proteins as possible. In 2010, however, funding for the PSI was terminated. Nevertheless, millions of protein structures were found, and many structures were uncharacterized. One of these uncharacterized proteins found in Chitinophaga pinesis. Its molecular weight is 87.1 kDa. Through structural and sequential analysis on BLAST, PFam, Dali, and ProMol, 4Q7Q is believed to be a lipase.<br />
==Sequential analysis of 4Q7Q==<br />
2.1 BLAST<br />
Sequence homology between 4Q7Q was tested through BLAST; the top hit for 4Q7Q was 4M8K, a GDSL-like lipase. 4Q7Q and 4M8k share a 36% sequence homology, but a low E-value, indicating it is a significant match. <br />
<br />
2.2 PFam<br />
Results from PFam sequence mapping also indicated that 4Q7Q shares sequence homology with other GDSL-like lipases. <br />
<br />
2.3 Snapgene<br />
Snapgene was able to reveal the molecular mass of 4Q7Q, later used as a reference when its purified form was run on an SDS-page gel. Snapgene also provided information about tags used to insert its gene into the plasmid used for transformation in DH5α and BL21 E. coli cells.<br />
== Structural analysis of 4Q7Q==<br />
3.1 Dali<br />
Top hits from Dali indicated that 4Q7Q shares structural homology with putative lipases.<br />
<br />
3.2 ProMol<br />
Through ProMol, a possible catalytic triad for 4Q7Q (Ser164, Asp193, His196) was found due to catalytic triad alignment with 1BRW. 4Q7Q and 1BRW catalytic triad alignment yielded a RMS value of 2.049, and a full structural homology with an RMS value of 4.852.<br />
<br />
==Enzymatic Assay of 4Q7Q==<br />
4.1 Reasoning<br />
Results obtained in silico strongly suggested that 4Q7Q was most likely to be hydrolase, and specifically a lipase. Thus, a lipase assay was run on 4Q7Q to confirm its enzymatic function. The lipase assay conducted was done through spectrophotometric methods. Purified 4Q7Q was mixed in solution with P-nitrophenyl butyrate. When nitrophenyl butyrate undergoes hydrolysis, its products are p-nitrophenol and butyrate. P-nitrophenol’s color becomes yellow when this reaction happens in solution.Thus, when a lipase catalyzes the hydrolysis of P-nitrophenol butyrate, a color change in solution will occur. A colorimeter was set at 430nm to detect light in the “yellow” range and thereby measure the change in color occuring when 4Q7Q catalyzed the hydrolysis reaction.<br />
<br />
4.2 Results<br />
<br />
=4.3 Conclusion=<br />
Due to 4Q7Q’s low concentration found on the SDS-page gel, 4Q7Q to pNPB ratio in solution was 10:1. A logarithmic-curve was present during the lipase assay conducted on 4Q7Q, indicating it successfully catalyzed the hydrolysis of a lipid. <br />
==Future directions==<br />
5.1 Cloning<br />
To confirm the suspected catalytic triad of 4Q7Q, site directed mutagenesis of 4Q7Qs catalytic triad was performed through PCR. Substitutions of residues were completed as followed: Ser164Ala, Asp193Ala, His196Ala. Following a successful cloning, a p-NPB lipase assay will be performed to see whether the new mutations on 4Q7Q deterred catalysis of the hydrolysis reaction of p-NPB.<br />
<br />
5.2 Enzyme Kinetics of 4Q7Q<br />
Further data of enzyme activity of 4Q7Q using our lipase assay at different concentrations of p-NPB can be performed to create a Lineweaver-Burk plot.<br />
<br />
5.3 Optimization of enzymatic activity<br />
Conditions for optimized enzyme activity can be tested by doing pH and temperature iterations of the p-NPB assay.<br />
<br />
<br />
<br />
<br />
</StructureSection><br />
== References and Notes ==<br />
<references/></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_7&diff=2904775User:Jennifer Taylor/Sandbox 72018-05-25T11:27:00Z<p>Jennifer Taylor: </p>
<hr />
<div>==About 4Q7Q:A Hydrolase==<br />
Here is a polar hydrophobic residue image of my protein, <scene name='78/787195/4q7q_polar_and_hydrophobic/1'>4Q7Q</scene>.<br />
<StructureSection load='4q7q' size='340' side='both' caption='Caption for this structure' scene=''><br />
This is a default text for your page '''Jennifer Taylor/Sandbox 7'''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br />
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br />
<br />
==1 What is 4Q7Q?==<br />
In 2000, the protein structure initiative (PSI) began with the goal of finding the three dimensional structure of as many proteins as possible. In 2010, however, funding for the PSI was terminated. Nevertheless, millions of protein structures were found, and many structures were uncharacterized. One of these uncharacterized proteins found in Chitinophaga pinesis. Its molecular weight is 87.1 kDa. Through structural and sequential analysis on BLAST, PFam, Dali, and ProMol, 4Q7Q is believed to be a lipase.<br />
==2 Sequential analysis of 4Q7Q==<br />
=2.1 BLAST=<br />
Sequence homology between 4Q7Q was tested through BLAST; the top hit for 4Q7Q was 4M8K, a GDSL-like lipase. 4Q7Q and 4M8k share a 36% sequence homology, but a low E-value, indicating it is a significant match. <br />
=2.2 PFam=<br />
Results from PFam sequence mapping also indicated that 4Q7Q shares sequence homology with other GDSL-like lipases. <br />
=2.3 Snapgene=<br />
Snapgene was able to reveal the molecular mass of 4Q7Q, later used as a reference when its purified form was run on an SDS-page gel. Snapgene also provided information about tags used to insert its gene into the plasmid used for transformation in DH5α and BL21 E. coli cells.<br />
==3 Structural analysis of 4Q7Q==<br />
=3.1 Dali=<br />
Top hits from Dali indicated that 4Q7Q shares structural homology with putative lipases.<br />
=3.2 ProMol=<br />
Through ProMol, a possible catalytic triad for 4Q7Q (Ser164, Asp193, His196) was found due to catalytic triad alignment with 1BRW. 4Q7Q and 1BRW catalytic triad alignment yielded a RMS value of 2.049, and a full structural homology with an RMS value of 4.852.<br />
==4 Enzymatic Assay of 4Q7Q==<br />
=4.1 Reasoning=<br />
Results obtained in silico strongly suggested that 4Q7Q was most likely to be hydrolase, and specifically a lipase. Thus, a lipase assay was run on 4Q7Q to confirm its enzymatic function. The lipase assay conducted was done through spectrophotometric methods. Purified 4Q7Q was mixed in solution with P-nitrophenyl butyrate. When nitrophenyl butyrate undergoes hydrolysis, its products are p-nitrophenol and butyrate. P-nitrophenol’s color becomes yellow when this reaction happens in solution.Thus, when a lipase catalyzes the hydrolysis of P-nitrophenol butyrate, a color change in solution will occur. A colorimeter was set at 430nm to detect light in the “yellow” range and thereby measure the change in color occuring when 4Q7Q catalyzed the hydrolysis reaction.<br />
=4.2 Results=<br />
<br />
=4.3 Conclusion=<br />
Due to 4Q7Q’s low concentration found on the SDS-page gel, 4Q7Q to pNPB ratio in solution was 10:1. A logarithmic-curve was present during the lipase assay conducted on 4Q7Q, indicating it successfully catalyzed the hydrolysis of a lipid. <br />
==5 Future directions==<br />
=5.1 Cloning=<br />
To confirm the suspected catalytic triad of 4Q7Q, site directed mutagenesis of 4Q7Qs catalytic triad was performed through PCR. Substitutions of residues were completed as followed: Ser164Ala, Asp193Ala, His196Ala. Following a successful cloning, a p-NPB lipase assay will be performed to see whether the new mutations on 4Q7Q deterred catalysis of the hydrolysis reaction of p-NPB.<br />
=5.2 Enzyme Kinetics of 4Q7Q=<br />
Further data of enzyme activity of 4Q7Q using our lipase assay at different concentrations of p-NPB can be performed to create a Lineweaver-Burk plot.<br />
=5.3 Optimization of enzymatic activity=<br />
Conditions for optimized enzyme activity can be tested by doing pH and temperature iterations of the p-NPB assay.<br />
<br />
<br />
<br />
<br />
</StructureSection><br />
== References and Notes ==<br />
<references/></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_1&diff=2904774User:Jennifer Taylor/Sandbox 12018-05-25T10:25:55Z<p>Jennifer Taylor: </p>
<hr />
<div>==YxiM from ''Bacillus subtilis''==<br />
<br />
<StructureSection load='2o14' size='340' side='right' caption='Structure of YxiM' scene=''><br />
<br />
== Background ==<br />
<br />
Proteins are an important type of macromolecule in biological systems and can be considered a sequence of subunits known as amino acids. The development of high-throughput genome squencing techniques allowed proteins to be sequenced more quickly than their structures could be solved. In an effort to close this gap, in 2000, the National Institutes of Health launched the 15-year Protein Structure Initiative. Many structures were deposited in the Protein Data Bank, but many of these proteins with solved structures, such as YxiM ([https://www.rcsb.org/structure/2o14 PDB ID: 2O14]), remain functionally uncharacterized. YxiM is transcribed by the ''yxiM'' gene from ''Bacillus subtilis'', a ubiquitous bacterial species that dwells in soil and gastrointestinal tracts. <scene name='78/787191/2o14_rainbow/2'>YxiM</scene> is 375 amino acids in length and its molecular weight is 41.8 kDa. It appears to have two domains: <scene name='78/787191/2o14_alpha/1'>one dominated by α-helices</scene>, and <scene name='78/787191/2o14_beta/1'>one by β-sheets</scene>.<br />
<br />
== ''In silico'' Analysis ==<br />
<br />
A common theme in biology is that form follows function. Thus, we used computer programs to find which proteins were most homologous to YxiM in terms of sequence and structure, with the expectation that YxiM is likely to be functionally similar to those proteins that have similar sequences and structures.<br />
<br />
[[Image:YxiM_green-1J00_blue.png|thumb|left|250px|Figure 1: insert your caption]]<br />
<br />
We used BLAST and PFam to find characterized proteins whose sequences aligned best with YxiM. Sequence analysis suggests that YxiM is a GDSL-like lipase, a type of esterase. Esterases are molecules that hydrolyze (decompose) a class of organic molecules known as esters. GDSL-like lipases demonstrate broad substrate specificity due to their flexible structures. BLAST showed that the proteins 1J00, 1IVN, and 1JRL have the highest sequence homology to YxiM. These proteins are multifunctional hydrolases that show both esterase and protease activity.<br />
<br />
Next, we used PyMOL to align the 3D structures of the BLAST hits with that of YxiM. The proteins 1J00, 1IVN, and 1JRL all align well with the α-helix domain of YxiM.<br />
<br />
[[Image:YxiM-1BWR_Catalytic.png|thumb|right|250px|Figure 2: insert your caption]]<br />
<br />
The Dali server finds the most similar proteins based on 3D structures, and the top 30 hits for YxiM were are all rhamnogalacturonan acetylesterases, GDSL lipases, LAE5s (hydrolases), or acetyl xylan esterases, which further suggests that YxiM is an esterase.<br />
<br />
Finally, we used ProMOL to perform a structural alignment of active sites of other proteins with YxiM to predict the active site of YxiM. We found that YxiM aligns best with the active site of IBWR, which is an esterase. The <scene name='78/787191/2o14_active_site/2'>putative catalytic triad</scene> of YxiM consists of amino acids S171, D339, and H342.<br />
<br />
Based on these analyses, we predicted that YxiM is an esterase and proceeded to perform ''in vitro'' assays to confirm esterase activity.<br />
<br />
== Plasmid Purification ==<br />
<br />
[[Image:PET21-YxiM_Map.png|thumb|right|250px|Figure 3: insert your caption]]<br />
<br />
In order to study the protein YxiM, we ordered a plasmid that contains the gene that transcribes the protein. A plasmid is a type of circular bacterial DNA. By transforming (inserting) this plasmid (pET21-''yxiM'') into the bacteria (DH5α Competent ''E. coli''), we can use the bacteria to create more of the plasmid. Then, we performed a DNA miniprep to purify the plasmid for later use.<br />
<br />
== Bacterial Transformation ==<br />
<br />
While DH5α ''E. coli'' are good for purifying plasmids, BL21(DE3) ''E. coli'' are more efficient for expressing protein. Thus, we transformed the plasmid into BL21(DE3) for the purposes of protein expression. We plated the bacteria on agar with the antibiotic ampicillin. While normal ''E. coli'' will die in the presence of ampicillin, the pET21-''yxiM'' plasmid has a gene that allows bacteria to become ampicillin resistant. Thus, only bacteria that were successfully transformed by the plasmid will survive on the ampicillin plate, allowing us to select for bacteria that have been transformed and thus bacteria that will express the protein YxiM.<br />
<br />
== Protein Expression ==<br />
<br />
After a day, colonies of transformed bacteria were visible on the agar plates. To express YxiM, we inoculated a single colony of bacteria into a liquid culture. In the plasmid, the ''yxiM'' gene is under control of the ''lac'' operon. This means that in the absence of an inducer, the transcription of the ''yxiM'' gene is repressed. Thus, we added IPTG, to activate the operon and drive the transcription of the protein YxiM.<br />
<br />
== Protein Purification ==<br />
<br />
After several hours, the bacteria have produced a relatively large amount of YxiM. To collect the protein, which at this point remained inside the cells, the bacteria were lysed (their cell walls were burst). The resulting mixture consisted of various cellular proteins and debris. The plasmid DNA sequence that coded for YxiM added a "tag" of histidines at the end of the protein. This allows us to separate YxiM proteins from the other types of proteins in the ''E. coli'' cells because when the mixture is passed through a nickel column, the tagged YxiM proteins stick to the column, while the other proteins flow through. Finally, we added elution buffer to the columns, which caused the proteins to detach from the nickel columns, creating a solution of just the YxiM proteins.<br />
<br />
== Esterase Activity Assay ==<br />
<br />
[[Image:YxiM_Lineweaver-Burk_Plot.png|thumb|left|250px|Figure 4: insert your caption]]<br />
<br />
Now that we had purified protein, we could test the function of YxiM ''in vitro''. Since we believed that YxiM was an ester, we placed it in a buffered solution with 4-nitrophenyl butyrate, a type of ester. Esterases should hydrolyze 4-nitrophenyl butyrate, causing the products butyric acid and 4-nitrophenol to form. Since 4-nitrophenyl is a yellow color, the absorbance of the solution changes as more products are formed. We used colorimeter to measure the absorbance at 430 nm as a proxy for esterase activity. We found that the absorbance increases over time, which suggests that YxiM is indeed an esterase.<br />
<br />
Specifically, we found that the Lineweaver-Burk plot of esterase activity is linear. This is typical of enzymes, as predicted by the Michaelis-Menten model of enzyme kinetics. Technically, we did not construct a true Lineweaver-Burk plot, as we used absorbance as a proxy for molar concentration, but absorbance varies linearly with concentration, as shown by the Beer-Lambert law.<br />
<br />
== Discussion ==<br />
<br />
YxiM is a previously uncharacterized protein whose crystal structure has been solved and deposited in the PDB.<br />
<br />
Protein sequence analysis with BLAST shows that YxiM is likely an esterase. PyMOL shows that the top BLAST hits also align well with the 3D structure of the α-helix domain of YxiM. Almost all the top structural hits in Dali are esterases as well, and ProMOL shows that the active site of YxiM most resembles one of an esterase or protease. The same catalytic triad (S171, D339, H342) is implicated in both protease and esterase activity, suggesting YxiM could be a multifunctional hydrolase. The catalytic motif of the esterase 1BWR aligns particularly well with YxiM.<br />
<br />
We tested YxiM for esterase activity ''in vitro'' in an effort to confirm the ''in silico'' predictions. YxiM showed esterase activity on 4-nitrophenyl butyrate, as absorbance increased during the assay. The Lineweaver-Burk plot of YxiM esterase activity is linear, which is typical of enzymes.<br />
<br />
Thus, on the basis of protein sequence and structural analysis ''in silico'' and functional assays ''in vitro'', we conclude that YxiM is an esterase.<br />
<br />
== Future Directions ==<br />
<br />
To further confirm the activity of YxiM as an esterase, we can perform mutagenesis on the putative catalytic triad by performing PCR on the plasmid DNA with specialized primers. If we mutate the catalytic triad, then we expect that the protein will not be able to perform its function anymore. Through another round of transformation and purification of this mutated DNA, we would expect the protein to show no activity in our esterase assay.<br />
<br />
Our analysis suggests that YxiM could also demonstrate protease activity. To test this, we could perform protease assays as well.<br />
<br />
To further study enzyme kinetics, we could relate absorbance with concentration of protein. We can achieve this by performing a Bradford protein assay to compute the extinction coefficient.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_1&diff=2904773User:Jennifer Taylor/Sandbox 12018-05-25T10:24:38Z<p>Jennifer Taylor: </p>
<hr />
<div>==YxiM from ''Bacillus subtilis''==<br />
<br />
<StructureSection load='2o14' size='340' side='right' caption='Structure of YxiM' scene=''><br />
<br />
== Background ==<br />
<br />
Proteins are an important type of macromolecule in biological systems and can be considered a sequence of subunits known as amino acids. The development of high-throughput genome squencing techniques allowed proteins to be sequenced more quickly than their structures could be solved. In an effort to close this gap, in 2000, the National Institutes of Health launched the 15-year Protein Structure Initiative. Many structures were deposited in the Protein Data Bank, but many of these proteins with solved structures, such as YxiM ([https://www.rcsb.org/structure/2o14 PDB ID: 2O14]), remain functionally uncharacterized. YxiM is transcribed by the ''yxiM'' gene from ''Bacillus subtilis'', a ubiquitous bacterial species that dwells in soil and gastrointestinal tracts. <scene name='78/787191/2o14_rainbow/2'>YxiM</scene> is 375 amino acids in length and its molecular weight is 41.8 kDa. It appears to have two domains: <scene name='78/787191/2o14_alpha/1'>one dominated by α-helices</scene>, and <scene name='78/787191/2o14_beta/1'>one by β-sheets</scene>.<br />
<br />
== ''In silico'' Analysis ==<br />
<br />
A common theme in biology is that form follows function. Thus, we used computer programs to find which proteins were most homologous to YxiM in terms of sequence and structure, with the expectation that YxiM is likely to be functionally similar to those proteins that have similar sequences and structures.<br />
<br />
[[Image:YxiM_green-1J00_blue.png|thumb|left|250px|Figure 1: insert your caption]]<br />
<br />
We used BLAST and PFam to find characterized proteins whose sequences aligned best with YxiM. Sequence analysis suggests that YxiM is a GDSL-like lipase, a type of esterase. Esterases are molecules that hydrolyze (decompose) a class of organic molecules known as esters. GDSL-like lipases demonstrate broad substrate specificity due to their flexible structures. BLAST showed that the proteins 1J00, 1IVN, and 1JRL have the highest sequence homology to YxiM. These proteins are multifunctional hydrolases that show both esterase and protease activity.<br />
<br />
Next, we used PyMOL to align the 3D structures of the BLAST hits with that of YxiM. The proteins 1J00, 1IVN, and 1JRL all align well with the α-helix domain of YxiM.<br />
<br />
[[Image:YxiM-1BWR_Catalytic.png|thumb|right|250px|Figure 2: insert your caption]]<br />
<br />
The Dali server finds the most similar proteins based on 3D structures, and the top 30 hits for YxiM were are all rhamnogalacturonan acetylesterases, GDSL lipases, LAE5s (hydrolases), or acetyl xylan esterases, which further suggests that YxiM is an esterase.<br />
<br />
Finally, we used ProMOL to perform a structural alignment of active sites of other proteins with YxiM to predict the active site of YxiM. We found that YxiM aligns best with the active site of IBWR, which is an esterase. The <scene name='78/787191/2o14_active_site/2'>putative catalytic triad</scene> of YxiM consists of amino acids S171, D339, and H342.<br />
<br />
Based on these analyses, we predicted that YxiM is an esterase and proceeded to perform ''in vitro'' assays to confirm esterase activity.<br />
<br />
== Plasmid Purification ==<br />
<br />
In order to study the protein YxiM, we ordered a plasmid that contains the gene that transcribes the protein. A plasmid is a type of circular bacterial DNA. By transforming (inserting) this plasmid (pET21-''yxiM'') into the bacteria (DH5α Competent ''E. coli''), we can use the bacteria to create more of the plasmid. Then, we performed a DNA miniprep to purify the plasmid for later use.<br />
<br />
[[Image:PET21-YxiM_Map.png|thumb|left|250px|Figure 3: insert your caption]]<br />
<br />
== Bacterial Transformation ==<br />
<br />
While DH5α ''E. coli'' are good for purifying plasmids, BL21(DE3) ''E. coli'' are more efficient for expressing protein. Thus, we transformed the plasmid into BL21(DE3) for the purposes of protein expression. We plated the bacteria on agar with the antibiotic ampicillin. While normal ''E. coli'' will die in the presence of ampicillin, the pET21-''yxiM'' plasmid has a gene that allows bacteria to become ampicillin resistant. Thus, only bacteria that were successfully transformed by the plasmid will survive on the ampicillin plate, allowing us to select for bacteria that have been transformed and thus bacteria that will express the protein YxiM.<br />
<br />
== Protein Expression ==<br />
<br />
After a day, colonies of transformed bacteria were visible on the agar plates. To express YxiM, we inoculated a single colony of bacteria into a liquid culture. In the plasmid, the ''yxiM'' gene is under control of the ''lac'' operon. This means that in the absence of an inducer, the transcription of the ''yxiM'' gene is repressed. Thus, we added IPTG, to activate the operon and drive the transcription of the protein YxiM.<br />
<br />
== Protein Purification ==<br />
<br />
After several hours, the bacteria have produced a relatively large amount of YxiM. To collect the protein, which at this point remained inside the cells, the bacteria were lysed (their cell walls were burst). The resulting mixture consisted of various cellular proteins and debris. The plasmid DNA sequence that coded for YxiM added a "tag" of histidines at the end of the protein. This allows us to separate YxiM proteins from the other types of proteins in the ''E. coli'' cells because when the mixture is passed through a nickel column, the tagged YxiM proteins stick to the column, while the other proteins flow through. Finally, we added elution buffer to the columns, which caused the proteins to detach from the nickel columns, creating a solution of just the YxiM proteins.<br />
<br />
== Esterase Activity Assay ==<br />
<br />
Now that we had purified protein, we could test the function of YxiM ''in vitro''. Since we believed that YxiM was an ester, we placed it in a buffered solution with 4-nitrophenyl butyrate, a type of ester. Esterases should hydrolyze 4-nitrophenyl butyrate, causing the products butyric acid and 4-nitrophenol to form. Since 4-nitrophenyl is a yellow color, the absorbance of the solution changes as more products are formed. We used colorimeter to measure the absorbance at 430 nm as a proxy for esterase activity. We found that the absorbance increases over time, which suggests that YxiM is indeed an esterase.<br />
<br />
Specifically, we found that the Lineweaver-Burk plot of esterase activity is linear. This is typical of enzymes, as predicted by the Michaelis-Menten model of enzyme kinetics. Technically, we did not construct a true Lineweaver-Burk plot, as we used absorbance as a proxy for molar concentration, but absorbance varies linearly with concentration, as shown by the Beer-Lambert law.<br />
<br />
[[Image:YxiM_Lineweaver-Burk_Plot.png|thumb|left|250px|Figure 4: insert your caption]]<br />
<br />
== Discussion ==<br />
<br />
YxiM is a previously uncharacterized protein whose crystal structure has been solved and deposited in the PDB.<br />
<br />
Protein sequence analysis with BLAST shows that YxiM is likely an esterase. PyMOL shows that the top BLAST hits also align well with the 3D structure of the α-helix domain of YxiM. Almost all the top structural hits in Dali are esterases as well, and ProMOL shows that the active site of YxiM most resembles one of an esterase or protease. The same catalytic triad (S171, D339, H342) is implicated in both protease and esterase activity, suggesting YxiM could be a multifunctional hydrolase. The catalytic motif of the esterase 1BWR aligns particularly well with YxiM.<br />
<br />
We tested YxiM for esterase activity ''in vitro'' in an effort to confirm the ''in silico'' predictions. YxiM showed esterase activity on 4-nitrophenyl butyrate, as absorbance increased during the assay. The Lineweaver-Burk plot of YxiM esterase activity is linear, which is typical of enzymes.<br />
<br />
Thus, on the basis of protein sequence and structural analysis ''in silico'' and functional assays ''in vitro'', we conclude that YxiM is an esterase.<br />
<br />
== Future Directions ==<br />
<br />
To further confirm the activity of YxiM as an esterase, we can perform mutagenesis on the putative catalytic triad by performing PCR on the plasmid DNA with specialized primers. If we mutate the catalytic triad, then we expect that the protein will not be able to perform its function anymore. Through another round of transformation and purification of this mutated DNA, we would expect the protein to show no activity in our esterase assay.<br />
<br />
Our analysis suggests that YxiM could also demonstrate protease activity. To test this, we could perform protease assays as well.<br />
<br />
To further study enzyme kinetics, we could relate absorbance with concentration of protein. We can achieve this by performing a Bradford protein assay to compute the extinction coefficient.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_1&diff=2904772User:Jennifer Taylor/Sandbox 12018-05-25T10:23:52Z<p>Jennifer Taylor: </p>
<hr />
<div>==YxiM from ''Bacillus subtilis''==<br />
<br />
<StructureSection load='2o14' size='340' side='right' caption='Structure of YxiM' scene=''><br />
<br />
== Background ==<br />
<br />
Proteins are an important type of macromolecule in biological systems and can be considered a sequence of subunits known as amino acids. The development of high-throughput genome squencing techniques allowed proteins to be sequenced more quickly than their structures could be solved. In an effort to close this gap, in 2000, the National Institutes of Health launched the 15-year Protein Structure Initiative. Many structures were deposited in the Protein Data Bank, but many of these proteins with solved structures, such as YxiM ([https://www.rcsb.org/structure/2o14 PDB ID: 2O14]), remain functionally uncharacterized. YxiM is transcribed by the ''yxiM'' gene from ''Bacillus subtilis'', a ubiquitous bacterial species that dwells in soil and gastrointestinal tracts. <scene name='78/787191/2o14_rainbow/2'>YxiM</scene> is 375 amino acids in length and its molecular weight is 41.8 kDa. It appears to have two domains: <scene name='78/787191/2o14_alpha/1'>one dominated by α-helices</scene>, and <scene name='78/787191/2o14_beta/1'>one by β-sheets</scene>.<br />
<br />
== ''In silico'' Analysis ==<br />
<br />
A common theme in biology is that form follows function. Thus, we used computer programs to find which proteins were most homologous to YxiM in terms of sequence and structure, with the expectation that YxiM is likely to be functionally similar to those proteins that have similar sequences and structures.<br />
<br />
We used BLAST and PFam to find characterized proteins whose sequences aligned best with YxiM. Sequence analysis suggests that YxiM is a GDSL-like lipase, a type of esterase. Esterases are molecules that hydrolyze (decompose) a class of organic molecules known as esters. GDSL-like lipases demonstrate broad substrate specificity due to their flexible structures. BLAST showed that the proteins 1J00, 1IVN, and 1JRL have the highest sequence homology to YxiM. These proteins are multifunctional hydrolases that show both esterase and protease activity.<br />
<br />
[[Image:YxiM_green-1J00_blue.png|thumb|left|250px|Figure 2: insert your caption]]<br />
<br />
Next, we used PyMOL to align the 3D structures of the BLAST hits with that of YxiM. The proteins 1J00, 1IVN, and 1JRL all align well with the α-helix domain of YxiM.<br />
<br />
The Dali server finds the most similar proteins based on 3D structures, and the top 30 hits for YxiM were are all rhamnogalacturonan acetylesterases, GDSL lipases, LAE5s (hydrolases), or acetyl xylan esterases, which further suggests that YxiM is an esterase.<br />
<br />
Finally, we used ProMOL to perform a structural alignment of active sites of other proteins with YxiM to predict the active site of YxiM. We found that YxiM aligns best with the active site of IBWR, which is an esterase. The <scene name='78/787191/2o14_active_site/2'>putative catalytic triad</scene> of YxiM consists of amino acids S171, D339, and H342.<br />
<br />
[[Image:YxiM-1BWR_Catalytic.png|thumb|right|250px|Figure 2: insert your caption]]<br />
<br />
Based on these analyses, we predicted that YxiM is an esterase and proceeded to perform ''in vitro'' assays to confirm esterase activity.<br />
<br />
== Plasmid Purification ==<br />
<br />
In order to study the protein YxiM, we ordered a plasmid that contains the gene that transcribes the protein. A plasmid is a type of circular bacterial DNA. By transforming (inserting) this plasmid (pET21-''yxiM'') into the bacteria (DH5α Competent ''E. coli''), we can use the bacteria to create more of the plasmid. Then, we performed a DNA miniprep to purify the plasmid for later use.<br />
<br />
[[Image:PET21-YxiM_Map.png|thumb|left|250px|Figure 1: insert your caption]]<br />
<br />
== Bacterial Transformation ==<br />
<br />
While DH5α ''E. coli'' are good for purifying plasmids, BL21(DE3) ''E. coli'' are more efficient for expressing protein. Thus, we transformed the plasmid into BL21(DE3) for the purposes of protein expression. We plated the bacteria on agar with the antibiotic ampicillin. While normal ''E. coli'' will die in the presence of ampicillin, the pET21-''yxiM'' plasmid has a gene that allows bacteria to become ampicillin resistant. Thus, only bacteria that were successfully transformed by the plasmid will survive on the ampicillin plate, allowing us to select for bacteria that have been transformed and thus bacteria that will express the protein YxiM.<br />
<br />
== Protein Expression ==<br />
<br />
After a day, colonies of transformed bacteria were visible on the agar plates. To express YxiM, we inoculated a single colony of bacteria into a liquid culture. In the plasmid, the ''yxiM'' gene is under control of the ''lac'' operon. This means that in the absence of an inducer, the transcription of the ''yxiM'' gene is repressed. Thus, we added IPTG, to activate the operon and drive the transcription of the protein YxiM.<br />
<br />
== Protein Purification ==<br />
<br />
After several hours, the bacteria have produced a relatively large amount of YxiM. To collect the protein, which at this point remained inside the cells, the bacteria were lysed (their cell walls were burst). The resulting mixture consisted of various cellular proteins and debris. The plasmid DNA sequence that coded for YxiM added a "tag" of histidines at the end of the protein. This allows us to separate YxiM proteins from the other types of proteins in the ''E. coli'' cells because when the mixture is passed through a nickel column, the tagged YxiM proteins stick to the column, while the other proteins flow through. Finally, we added elution buffer to the columns, which caused the proteins to detach from the nickel columns, creating a solution of just the YxiM proteins.<br />
<br />
== Esterase Activity Assay ==<br />
<br />
Now that we had purified protein, we could test the function of YxiM ''in vitro''. Since we believed that YxiM was an ester, we placed it in a buffered solution with 4-nitrophenyl butyrate, a type of ester. Esterases should hydrolyze 4-nitrophenyl butyrate, causing the products butyric acid and 4-nitrophenol to form. Since 4-nitrophenyl is a yellow color, the absorbance of the solution changes as more products are formed. We used colorimeter to measure the absorbance at 430 nm as a proxy for esterase activity. We found that the absorbance increases over time, which suggests that YxiM is indeed an esterase.<br />
<br />
Specifically, we found that the Lineweaver-Burk plot of esterase activity is linear. This is typical of enzymes, as predicted by the Michaelis-Menten model of enzyme kinetics. Technically, we did not construct a true Lineweaver-Burk plot, as we used absorbance as a proxy for molar concentration, but absorbance varies linearly with concentration, as shown by the Beer-Lambert law.<br />
<br />
[[Image:YxiM_Lineweaver-Burk_Plot.png|thumb|left|250px|Figure 2: insert your caption]]<br />
<br />
== Discussion ==<br />
<br />
YxiM is a previously uncharacterized protein whose crystal structure has been solved and deposited in the PDB.<br />
<br />
Protein sequence analysis with BLAST shows that YxiM is likely an esterase. PyMOL shows that the top BLAST hits also align well with the 3D structure of the α-helix domain of YxiM. Almost all the top structural hits in Dali are esterases as well, and ProMOL shows that the active site of YxiM most resembles one of an esterase or protease. The same catalytic triad (S171, D339, H342) is implicated in both protease and esterase activity, suggesting YxiM could be a multifunctional hydrolase. The catalytic motif of the esterase 1BWR aligns particularly well with YxiM.<br />
<br />
We tested YxiM for esterase activity ''in vitro'' in an effort to confirm the ''in silico'' predictions. YxiM showed esterase activity on 4-nitrophenyl butyrate, as absorbance increased during the assay. The Lineweaver-Burk plot of YxiM esterase activity is linear, which is typical of enzymes.<br />
<br />
Thus, on the basis of protein sequence and structural analysis ''in silico'' and functional assays ''in vitro'', we conclude that YxiM is an esterase.<br />
<br />
== Future Directions ==<br />
<br />
To further confirm the activity of YxiM as an esterase, we can perform mutagenesis on the putative catalytic triad by performing PCR on the plasmid DNA with specialized primers. If we mutate the catalytic triad, then we expect that the protein will not be able to perform its function anymore. Through another round of transformation and purification of this mutated DNA, we would expect the protein to show no activity in our esterase assay.<br />
<br />
Our analysis suggests that YxiM could also demonstrate protease activity. To test this, we could perform protease assays as well.<br />
<br />
To further study enzyme kinetics, we could relate absorbance with concentration of protein. We can achieve this by performing a Bradford protein assay to compute the extinction coefficient.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_1&diff=2904771User:Jennifer Taylor/Sandbox 12018-05-25T10:15:25Z<p>Jennifer Taylor: </p>
<hr />
<div>==YxiM from ''Bacillus subtilis''==<br />
<br />
<StructureSection load='2o14' size='340' side='right' caption='Structure of YxiM' scene=''><br />
<br />
== Background ==<br />
<br />
Proteins are an important type of macromolecule in biological systems and can be considered a sequence of subunits known as amino acids. The development of high-throughput genome squencing techniques allowed proteins to be sequenced more quickly than their structures could be solved. In an effort to close this gap, in 2000, the National Institutes of Health launched the 15-year Protein Structure Initiative. Many structures were deposited in the Protein Data Bank, but many of these proteins with solved structures, such as YxiM ([https://www.rcsb.org/structure/2o14 PDB ID: 2O14]), remain functionally uncharacterized. YxiM is transcribed by the ''yxiM'' gene from ''Bacillus subtilis'', a ubiquitous bacterial species that dwells in soil and gastrointestinal tracts. <scene name='78/787191/2o14_rainbow/2'>YxiM</scene> is 375 amino acids in length and its molecular weight is 41.8 kDa. It appears to have two domains: <scene name='78/787191/2o14_alpha/1'>one dominated by α-helices</scene>, and <scene name='78/787191/2o14_beta/1'>one by β-sheets</scene>.<br />
<br />
== ''In silico'' Analysis ==<br />
<br />
A common theme in biology is that form follows function. Thus, we used computer programs to find which proteins were most homologous to YxiM in terms of sequence and structure, with the expectation that YxiM is likely to be functionally similar to those proteins that have similar sequences and structures.<br />
<br />
We used BLAST and PFam to find characterized proteins whose sequences aligned best with YxiM. Sequence analysis suggests that YxiM is a GDSL-like lipase, a type of esterase. Esterases are molecules that hydrolyze (decompose) a class of organic molecules known as esters. GDSL-like lipases demonstrate broad substrate specificity due to their flexible structures. BLAST showed that the proteins 1J00, 1IVN, and 1JRL have the highest sequence homology to YxiM. These proteins are multifunctional hydrolases that show both esterase and protease activity.<br />
<br />
Next, we used PyMOL to align the 3D structures of the BLAST hits with that of YxiM. The proteins 1J00, 1IVN, and 1JRL all align well with the α-helix domain of YxiM.<br />
<br />
[[Image:YxiM_green-1J00_blue.png|thumb|left|250px|Figure 2: insert your caption]]<br />
<br />
The Dali server finds the most similar proteins based on 3D structures, and the top 30 hits for YxiM were are all rhamnogalacturonan acetylesterases, GDSL lipases, LAE5s (hydrolases), or acetyl xylan esterases, which further suggests that YxiM is an esterase.<br />
<br />
Finally, we used ProMOL to perform a structural alignment of active sites of other proteins with YxiM to predict the active site of YxiM. We found that YxiM aligns best with the active site of IBWR, which is an esterase. The <scene name='78/787191/2o14_active_site/2'>putative catalytic triad</scene> of YxiM consists of amino acids S171, D339, and H342.<br />
<br />
[[Image:YxiM-1BWR_Catalytic.png|thumb|left|250px|Figure 2: insert your caption]]<br />
<br />
Based on these analyses, we predicted that YxiM is an esterase and proceeded to perform ''in vitro'' assays to confirm esterase activity.<br />
<br />
== Plasmid Purification ==<br />
<br />
In order to study the protein YxiM, we ordered a plasmid that contains the gene that transcribes the protein. A plasmid is a type of circular bacterial DNA. By transforming (inserting) this plasmid (pET21-''yxiM'') into the bacteria (DH5α Competent ''E. coli''), we can use the bacteria to create more of the plasmid. Then, we performed a DNA miniprep to purify the plasmid for later use.<br />
<br />
[[Image:PET21-YxiM_Map.png|thumb|left|250px|Figure 1: insert your caption]]<br />
<br />
== Bacterial Transformation ==<br />
<br />
While DH5α ''E. coli'' are good for purifying plasmids, BL21(DE3) ''E. coli'' are more efficient for expressing protein. Thus, we transformed the plasmid into BL21(DE3) for the purposes of protein expression. We plated the bacteria on agar with the antibiotic ampicillin. While normal ''E. coli'' will die in the presence of ampicillin, the pET21-''yxiM'' plasmid has a gene that allows bacteria to become ampicillin resistant. Thus, only bacteria that were successfully transformed by the plasmid will survive on the ampicillin plate, allowing us to select for bacteria that have been transformed and thus bacteria that will express the protein YxiM.<br />
<br />
== Protein Expression ==<br />
<br />
After a day, colonies of transformed bacteria were visible on the agar plates. To express YxiM, we inoculated a single colony of bacteria into a liquid culture. In the plasmid, the ''yxiM'' gene is under control of the ''lac'' operon. This means that in the absence of an inducer, the transcription of the ''yxiM'' gene is repressed. Thus, we added IPTG, to activate the operon and drive the transcription of the protein YxiM.<br />
<br />
== Protein Purification ==<br />
<br />
After several hours, the bacteria have produced a relatively large amount of YxiM. To collect the protein, which at this point remained inside the cells, the bacteria were lysed (their cell walls were burst). The resulting mixture consisted of various cellular proteins and debris. The plasmid DNA sequence that coded for YxiM added a "tag" of histidines at the end of the protein. This allows us to separate YxiM proteins from the other types of proteins in the ''E. coli'' cells because when the mixture is passed through a nickel column, the tagged YxiM proteins stick to the column, while the other proteins flow through. Finally, we added elution buffer to the columns, which caused the proteins to detach from the nickel columns, creating a solution of just the YxiM proteins.<br />
<br />
== Esterase Activity Assay ==<br />
<br />
Now that we had purified protein, we could test the function of YxiM ''in vitro''. Since we believed that YxiM was an ester, we placed it in a buffered solution with 4-nitrophenyl butyrate, a type of ester. Esterases should hydrolyze 4-nitrophenyl butyrate, causing the products butyric acid and 4-nitrophenol to form. Since 4-nitrophenyl is a yellow color, the absorbance of the solution changes as more products are formed. We used colorimeter to measure the absorbance at 430 nm as a proxy for esterase activity. We found that the absorbance increases over time, which suggests that YxiM is indeed an esterase.<br />
<br />
Specifically, we found that the Lineweaver-Burk plot of esterase activity is linear. This is typical of enzymes, as predicted by the Michaelis-Menten model of enzyme kinetics. Technically, we did not construct a true Lineweaver-Burk plot, as we used absorbance as a proxy for molar concentration, but absorbance varies linearly with concentration, as shown by the Beer-Lambert law.<br />
<br />
[[Image:YxiM_Lineweaver-Burk_Plot.png|thumb|left|250px|Figure 2: insert your caption]]<br />
<br />
== Discussion ==<br />
<br />
YxiM is a previously uncharacterized protein whose crystal structure has been solved and deposited in the PDB.<br />
<br />
Protein sequence analysis with BLAST shows that YxiM is likely an esterase. PyMOL shows that the top BLAST hits also align well with the 3D structure of the α-helix domain of YxiM. Almost all the top structural hits in Dali are esterases as well, and ProMOL shows that the active site of YxiM most resembles one of an esterase or protease. The same catalytic triad (S171, D339, H342) is implicated in both protease and esterase activity, suggesting YxiM could be a multifunctional hydrolase. The catalytic motif of the esterase 1BWR aligns particularly well with YxiM.<br />
<br />
We tested YxiM for esterase activity ''in vitro'' in an effort to confirm the ''in silico'' predictions. YxiM showed esterase activity on 4-nitrophenyl butyrate, as absorbance increased during the assay. The Lineweaver-Burk plot of YxiM esterase activity is linear, which is typical of enzymes.<br />
<br />
Thus, on the basis of protein sequence and structural analysis ''in silico'' and functional assays ''in vitro'', we conclude that YxiM is an esterase.<br />
<br />
== Future Directions ==<br />
<br />
To further confirm the activity of YxiM as an esterase, we can perform mutagenesis on the putative catalytic triad by performing PCR on the plasmid DNA with specialized primers. If we mutate the catalytic triad, then we expect that the protein will not be able to perform its function anymore. Through another round of transformation and purification of this mutated DNA, we would expect the protein to show no activity in our esterase assay.<br />
<br />
Our analysis suggests that YxiM could also demonstrate protease activity. To test this, we could perform protease assays as well.<br />
<br />
To further study enzyme kinetics, we could relate absorbance with concentration of protein. We can achieve this by performing a Bradford protein assay to compute the extinction coefficient.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_1&diff=2904770User:Jennifer Taylor/Sandbox 12018-05-25T10:15:05Z<p>Jennifer Taylor: </p>
<hr />
<div>==YxiM from ''Bacillus subtilis''==<br />
<br />
<StructureSection load='2o14' size='340' side='right' caption='Structure of YxiM' scene=''><br />
<br />
== Background ==<br />
<br />
Proteins are an important type of macromolecule in biological systems and can be considered a sequence of subunits known as amino acids. The development of high-throughput genome squencing techniques allowed proteins to be sequenced more quickly than their structures could be solved. In an effort to close this gap, in 2000, the National Institutes of Health launched the 15-year Protein Structure Initiative. Many structures were deposited in the Protein Data Bank, but many of these proteins with solved structures, such as YxiM ([https://www.rcsb.org/structure/2o14 PDB ID: 2O14]), remain functionally uncharacterized. YxiM is transcribed by the ''yxiM'' gene from ''Bacillus subtilis'', a ubiquitous bacterial species that dwells in soil and gastrointestinal tracts. <scene name='78/787191/2o14_rainbow/2'>YxiM</scene> is 375 amino acids in length and its molecular weight is 41.8 kDa. It appears to have two domains: <scene name='78/787191/2o14_alpha/1'>one dominated by α-helices</scene>, and <scene name='78/787191/2o14_beta/1'>one by β-sheets</scene>.<br />
<br />
== ''In silico'' Analysis ==<br />
<br />
A common theme in biology is that form follows function. Thus, we used computer programs to find which proteins were most homologous to YxiM in terms of sequence and structure, with the expectation that YxiM is likely to be functionally similar to those proteins that have similar sequences and structures.<br />
<br />
We used BLAST and PFam to find characterized proteins whose sequences aligned best with YxiM. Sequence analysis suggests that YxiM is a GDSL-like lipase, a type of esterase. Esterases are molecules that hydrolyze (decompose) a class of organic molecules known as esters. GDSL-like lipases demonstrate broad substrate specificity due to their flexible structures. BLAST showed that the proteins 1J00, 1IVN, and 1JRL have the highest sequence homology to YxiM. These proteins are multifunctional hydrolases that show both esterase and protease activity.<br />
<br />
Next, we used PyMOL to align the 3D structures of the BLAST hits with that of YxiM. The proteins 1J00, 1IVN, and 1JRL all align well with the α-helix domain of YxiM.<br />
<br />
[[Image: YxiM_green-1J00_blue.png|thumb|left|250px|Figure 2: insert your caption]]<br />
<br />
The Dali server finds the most similar proteins based on 3D structures, and the top 30 hits for YxiM were are all rhamnogalacturonan acetylesterases, GDSL lipases, LAE5s (hydrolases), or acetyl xylan esterases, which further suggests that YxiM is an esterase.<br />
<br />
Finally, we used ProMOL to perform a structural alignment of active sites of other proteins with YxiM to predict the active site of YxiM. We found that YxiM aligns best with the active site of IBWR, which is an esterase. The <scene name='78/787191/2o14_active_site/2'>putative catalytic triad</scene> of YxiM consists of amino acids S171, D339, and H342.<br />
<br />
[[Image:YxiM-1BWR_Catalytic.png|thumb|left|250px|Figure 2: insert your caption]]<br />
<br />
Based on these analyses, we predicted that YxiM is an esterase and proceeded to perform ''in vitro'' assays to confirm esterase activity.<br />
<br />
== Plasmid Purification ==<br />
<br />
In order to study the protein YxiM, we ordered a plasmid that contains the gene that transcribes the protein. A plasmid is a type of circular bacterial DNA. By transforming (inserting) this plasmid (pET21-''yxiM'') into the bacteria (DH5α Competent ''E. coli''), we can use the bacteria to create more of the plasmid. Then, we performed a DNA miniprep to purify the plasmid for later use.<br />
<br />
[[Image:PET21-YxiM_Map.png|thumb|left|250px|Figure 1: insert your caption]]<br />
<br />
== Bacterial Transformation ==<br />
<br />
While DH5α ''E. coli'' are good for purifying plasmids, BL21(DE3) ''E. coli'' are more efficient for expressing protein. Thus, we transformed the plasmid into BL21(DE3) for the purposes of protein expression. We plated the bacteria on agar with the antibiotic ampicillin. While normal ''E. coli'' will die in the presence of ampicillin, the pET21-''yxiM'' plasmid has a gene that allows bacteria to become ampicillin resistant. Thus, only bacteria that were successfully transformed by the plasmid will survive on the ampicillin plate, allowing us to select for bacteria that have been transformed and thus bacteria that will express the protein YxiM.<br />
<br />
== Protein Expression ==<br />
<br />
After a day, colonies of transformed bacteria were visible on the agar plates. To express YxiM, we inoculated a single colony of bacteria into a liquid culture. In the plasmid, the ''yxiM'' gene is under control of the ''lac'' operon. This means that in the absence of an inducer, the transcription of the ''yxiM'' gene is repressed. Thus, we added IPTG, to activate the operon and drive the transcription of the protein YxiM.<br />
<br />
== Protein Purification ==<br />
<br />
After several hours, the bacteria have produced a relatively large amount of YxiM. To collect the protein, which at this point remained inside the cells, the bacteria were lysed (their cell walls were burst). The resulting mixture consisted of various cellular proteins and debris. The plasmid DNA sequence that coded for YxiM added a "tag" of histidines at the end of the protein. This allows us to separate YxiM proteins from the other types of proteins in the ''E. coli'' cells because when the mixture is passed through a nickel column, the tagged YxiM proteins stick to the column, while the other proteins flow through. Finally, we added elution buffer to the columns, which caused the proteins to detach from the nickel columns, creating a solution of just the YxiM proteins.<br />
<br />
== Esterase Activity Assay ==<br />
<br />
Now that we had purified protein, we could test the function of YxiM ''in vitro''. Since we believed that YxiM was an ester, we placed it in a buffered solution with 4-nitrophenyl butyrate, a type of ester. Esterases should hydrolyze 4-nitrophenyl butyrate, causing the products butyric acid and 4-nitrophenol to form. Since 4-nitrophenyl is a yellow color, the absorbance of the solution changes as more products are formed. We used colorimeter to measure the absorbance at 430 nm as a proxy for esterase activity. We found that the absorbance increases over time, which suggests that YxiM is indeed an esterase.<br />
<br />
Specifically, we found that the Lineweaver-Burk plot of esterase activity is linear. This is typical of enzymes, as predicted by the Michaelis-Menten model of enzyme kinetics. Technically, we did not construct a true Lineweaver-Burk plot, as we used absorbance as a proxy for molar concentration, but absorbance varies linearly with concentration, as shown by the Beer-Lambert law.<br />
<br />
[[Image:YxiM_Lineweaver-Burk_Plot.png|thumb|left|250px|Figure 2: insert your caption]]<br />
<br />
== Discussion ==<br />
<br />
YxiM is a previously uncharacterized protein whose crystal structure has been solved and deposited in the PDB.<br />
<br />
Protein sequence analysis with BLAST shows that YxiM is likely an esterase. PyMOL shows that the top BLAST hits also align well with the 3D structure of the α-helix domain of YxiM. Almost all the top structural hits in Dali are esterases as well, and ProMOL shows that the active site of YxiM most resembles one of an esterase or protease. The same catalytic triad (S171, D339, H342) is implicated in both protease and esterase activity, suggesting YxiM could be a multifunctional hydrolase. The catalytic motif of the esterase 1BWR aligns particularly well with YxiM.<br />
<br />
We tested YxiM for esterase activity ''in vitro'' in an effort to confirm the ''in silico'' predictions. YxiM showed esterase activity on 4-nitrophenyl butyrate, as absorbance increased during the assay. The Lineweaver-Burk plot of YxiM esterase activity is linear, which is typical of enzymes.<br />
<br />
Thus, on the basis of protein sequence and structural analysis ''in silico'' and functional assays ''in vitro'', we conclude that YxiM is an esterase.<br />
<br />
== Future Directions ==<br />
<br />
To further confirm the activity of YxiM as an esterase, we can perform mutagenesis on the putative catalytic triad by performing PCR on the plasmid DNA with specialized primers. If we mutate the catalytic triad, then we expect that the protein will not be able to perform its function anymore. Through another round of transformation and purification of this mutated DNA, we would expect the protein to show no activity in our esterase assay.<br />
<br />
Our analysis suggests that YxiM could also demonstrate protease activity. To test this, we could perform protease assays as well.<br />
<br />
To further study enzyme kinetics, we could relate absorbance with concentration of protein. We can achieve this by performing a Bradford protein assay to compute the extinction coefficient.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_1&diff=2904769User:Jennifer Taylor/Sandbox 12018-05-25T10:13:43Z<p>Jennifer Taylor: </p>
<hr />
<div>==YxiM from ''Bacillus subtilis''==<br />
<br />
<StructureSection load='2o14' size='340' side='right' caption='Structure of YxiM' scene=''><br />
<br />
== Background ==<br />
<br />
Proteins are an important type of macromolecule in biological systems and can be considered a sequence of subunits known as amino acids. The development of high-throughput genome squencing techniques allowed proteins to be sequenced more quickly than their structures could be solved. In an effort to close this gap, in 2000, the National Institutes of Health launched the 15-year Protein Structure Initiative. Many structures were deposited in the Protein Data Bank, but many of these proteins with solved structures, such as YxiM ([https://www.rcsb.org/structure/2o14 PDB ID: 2O14]), remain functionally uncharacterized. YxiM is transcribed by the ''yxiM'' gene from ''Bacillus subtilis'', a ubiquitous bacterial species that dwells in soil and gastrointestinal tracts. <scene name='78/787191/2o14_rainbow/2'>YxiM</scene> is 375 amino acids in length and its molecular weight is 41.8 kDa. It appears to have two domains: <scene name='78/787191/2o14_alpha/1'>one dominated by α-helices</scene>, and <scene name='78/787191/2o14_beta/1'>one by β-sheets</scene>.<br />
<br />
== ''In silico'' Analysis ==<br />
<br />
A common theme in biology is that form follows function. Thus, we used computer programs to find which proteins were most homologous to YxiM in terms of sequence and structure, with the expectation that YxiM is likely to be functionally similar to those proteins that have similar sequences and structures.<br />
<br />
We used BLAST and PFam to find characterized proteins whose sequences aligned best with YxiM. Sequence analysis suggests that YxiM is a GDSL-like lipase, a type of esterase. Esterases are molecules that hydrolyze (decompose) a class of organic molecules known as esters. GDSL-like lipases demonstrate broad substrate specificity due to their flexible structures. BLAST showed that the proteins 1J00, 1IVN, and 1JRL have the highest sequence homology to YxiM. These proteins are multifunctional hydrolases that show both esterase and protease activity.<br />
<br />
Next, we used PyMOL to align the 3D structures of the BLAST hits with that of YxiM. The proteins 1J00, 1IVN, and 1JRL all align well with the α-helix domain of YxiM.<br />
<br />
The Dali server finds the most similar proteins based on 3D structures, and the top 30 hits for YxiM were are all rhamnogalacturonan acetylesterases, GDSL lipases, LAE5s (hydrolases), or acetyl xylan esterases, which further suggests that YxiM is an esterase.<br />
<br />
Finally, we used ProMOL to perform a structural alignment of active sites of other proteins with YxiM to predict the active site of YxiM. We found that YxiM aligns best with the active site of IBWR, which is an esterase. The <scene name='78/787191/2o14_active_site/2'>putative catalytic triad</scene> of YxiM consists of amino acids S171, D339, and H342.<br />
<br />
Based on these analyses, we predicted that YxiM is an esterase and proceeded to perform ''in vitro'' assays to confirm esterase activity.<br />
<br />
== Plasmid Purification ==<br />
<br />
In order to study the protein YxiM, we ordered a plasmid that contains the gene that transcribes the protein. A plasmid is a type of circular bacterial DNA. By transforming (inserting) this plasmid (pET21-''yxiM'') into the bacteria (DH5α Competent ''E. coli''), we can use the bacteria to create more of the plasmid. Then, we performed a DNA miniprep to purify the plasmid for later use.<br />
<br />
[[Image:PET21-YxiM_Map.png|thumb|left|250px|Figure 1: insert your caption]]<br />
<br />
== Bacterial Transformation ==<br />
<br />
While DH5α ''E. coli'' are good for purifying plasmids, BL21(DE3) ''E. coli'' are more efficient for expressing protein. Thus, we transformed the plasmid into BL21(DE3) for the purposes of protein expression. We plated the bacteria on agar with the antibiotic ampicillin. While normal ''E. coli'' will die in the presence of ampicillin, the pET21-''yxiM'' plasmid has a gene that allows bacteria to become ampicillin resistant. Thus, only bacteria that were successfully transformed by the plasmid will survive on the ampicillin plate, allowing us to select for bacteria that have been transformed and thus bacteria that will express the protein YxiM.<br />
<br />
== Protein Expression ==<br />
<br />
After a day, colonies of transformed bacteria were visible on the agar plates. To express YxiM, we inoculated a single colony of bacteria into a liquid culture. In the plasmid, the ''yxiM'' gene is under control of the ''lac'' operon. This means that in the absence of an inducer, the transcription of the ''yxiM'' gene is repressed. Thus, we added IPTG, to activate the operon and drive the transcription of the protein YxiM.<br />
<br />
== Protein Purification ==<br />
<br />
After several hours, the bacteria have produced a relatively large amount of YxiM. To collect the protein, which at this point remained inside the cells, the bacteria were lysed (their cell walls were burst). The resulting mixture consisted of various cellular proteins and debris. The plasmid DNA sequence that coded for YxiM added a "tag" of histidines at the end of the protein. This allows us to separate YxiM proteins from the other types of proteins in the ''E. coli'' cells because when the mixture is passed through a nickel column, the tagged YxiM proteins stick to the column, while the other proteins flow through. Finally, we added elution buffer to the columns, which caused the proteins to detach from the nickel columns, creating a solution of just the YxiM proteins.<br />
<br />
== Esterase Activity Assay ==<br />
<br />
Now that we had purified protein, we could test the function of YxiM ''in vitro''. Since we believed that YxiM was an ester, we placed it in a buffered solution with 4-nitrophenyl butyrate, a type of ester. Esterases should hydrolyze 4-nitrophenyl butyrate, causing the products butyric acid and 4-nitrophenol to form. Since 4-nitrophenyl is a yellow color, the absorbance of the solution changes as more products are formed. We used colorimeter to measure the absorbance at 430 nm as a proxy for esterase activity. We found that the absorbance increases over time, which suggests that YxiM is indeed an esterase.<br />
<br />
Specifically, we found that the Lineweaver-Burk plot of esterase activity is linear. This is typical of enzymes, as predicted by the Michaelis-Menten model of enzyme kinetics. Technically, we did not construct a true Lineweaver-Burk plot, as we used absorbance as a proxy for molar concentration, but absorbance varies linearly with concentration, as shown by the Beer-Lambert law.<br />
<br />
[[Image:YxiM_Lineweaver-Burk_Plot.png|thumb|left|250px|Figure 2: insert your caption]]<br />
<br />
== Discussion ==<br />
<br />
YxiM is a previously uncharacterized protein whose crystal structure has been solved and deposited in the PDB.<br />
<br />
Protein sequence analysis with BLAST shows that YxiM is likely an esterase. PyMOL shows that the top BLAST hits also align well with the 3D structure of the α-helix domain of YxiM. Almost all the top structural hits in Dali are esterases as well, and ProMOL shows that the active site of YxiM most resembles one of an esterase or protease. The same catalytic triad (S171, D339, H342) is implicated in both protease and esterase activity, suggesting YxiM could be a multifunctional hydrolase. The catalytic motif of the esterase 1BWR aligns particularly well with YxiM.<br />
<br />
We tested YxiM for esterase activity ''in vitro'' in an effort to confirm the ''in silico'' predictions. YxiM showed esterase activity on 4-nitrophenyl butyrate, as absorbance increased during the assay. The Lineweaver-Burk plot of YxiM esterase activity is linear, which is typical of enzymes.<br />
<br />
Thus, on the basis of protein sequence and structural analysis ''in silico'' and functional assays ''in vitro'', we conclude that YxiM is an esterase.<br />
<br />
== Future Directions ==<br />
<br />
To further confirm the activity of YxiM as an esterase, we can perform mutagenesis on the putative catalytic triad by performing PCR on the plasmid DNA with specialized primers. If we mutate the catalytic triad, then we expect that the protein will not be able to perform its function anymore. Through another round of transformation and purification of this mutated DNA, we would expect the protein to show no activity in our esterase assay.<br />
<br />
Our analysis suggests that YxiM could also demonstrate protease activity. To test this, we could perform protease assays as well.<br />
<br />
To further study enzyme kinetics, we could relate absorbance with concentration of protein. We can achieve this by performing a Bradford protein assay to compute the extinction coefficient.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=User:Jennifer_Taylor/Sandbox_1&diff=2904768User:Jennifer Taylor/Sandbox 12018-05-25T10:12:55Z<p>Jennifer Taylor: </p>
<hr />
<div>==YxiM from ''Bacillus subtilis''==<br />
<br />
<StructureSection load='2o14' size='340' side='right' caption='Structure of YxiM' scene=''><br />
<br />
== Background ==<br />
<br />
Proteins are an important type of macromolecule in biological systems and can be considered a sequence of subunits known as amino acids. The development of high-throughput genome squencing techniques allowed proteins to be sequenced more quickly than their structures could be solved. In an effort to close this gap, in 2000, the National Institutes of Health launched the 15-year Protein Structure Initiative. Many structures were deposited in the Protein Data Bank, but many of these proteins with solved structures, such as YxiM ([https://www.rcsb.org/structure/2o14 PDB ID: 2O14]), remain functionally uncharacterized. YxiM is transcribed by the ''yxiM'' gene from ''Bacillus subtilis'', a ubiquitous bacterial species that dwells in soil and gastrointestinal tracts. <scene name='78/787191/2o14_rainbow/2'>YxiM</scene> is 375 amino acids in length and its molecular weight is 41.8 kDa. It appears to have two domains: <scene name='78/787191/2o14_alpha/1'>one dominated by α-helices</scene>, and <scene name='78/787191/2o14_beta/1'>one by β-sheets</scene>.<br />
<br />
== ''In silico'' Analysis ==<br />
<br />
A common theme in biology is that form follows function. Thus, we used computer programs to find which proteins were most homologous to YxiM in terms of sequence and structure, with the expectation that YxiM is likely to be functionally similar to those proteins that have similar sequences and structures.<br />
<br />
We used BLAST and PFam to find characterized proteins whose sequences aligned best with YxiM. Sequence analysis suggests that YxiM is a GDSL-like lipase, a type of esterase. Esterases are molecules that hydrolyze (decompose) a class of organic molecules known as esters. GDSL-like lipases demonstrate broad substrate specificity due to their flexible structures. BLAST showed that the proteins 1J00, 1IVN, and 1JRL have the highest sequence homology to YxiM. These proteins are multifunctional hydrolases that show both esterase and protease activity.<br />
<br />
Next, we used PyMOL to align the 3D structures of the BLAST hits with that of YxiM. The proteins 1J00, 1IVN, and 1JRL all align well with the α-helix domain of YxiM.<br />
<br />
The Dali server finds the most similar proteins based on 3D structures, and the top 30 hits for YxiM were are all rhamnogalacturonan acetylesterases, GDSL lipases, LAE5s (hydrolases), or acetyl xylan esterases, which further suggests that YxiM is an esterase.<br />
<br />
Finally, we used ProMOL to perform a structural alignment of active sites of other proteins with YxiM to predict the active site of YxiM. We found that YxiM aligns best with the active site of IBWR, which is an esterase. The <scene name='78/787191/2o14_active_site/2'>putative catalytic triad</scene> of YxiM consists of amino acids S171, D339, and H342.<br />
<br />
Based on these analyses, we predicted that YxiM is an esterase and proceeded to perform ''in vitro'' assays to confirm esterase activity.<br />
<br />
== Plasmid Purification ==<br />
<br />
In order to study the protein YxiM, we ordered a plasmid that contains the gene that transcribes the protein. A plasmid is a type of circular bacterial DNA. By transforming (inserting) this plasmid (pET21-''yxiM'') into the bacteria (DH5α Competent ''E. coli''), we can use the bacteria to create more of the plasmid. Then, we performed a DNA miniprep to purify the plasmid for later use.<br />
<br />
[[Image:PET21-YxiM_Map.png|thumb|left|250px|Figure 1: insert your caption]]<br />
<br />
== Bacterial Transformation ==<br />
<br />
While DH5α ''E. coli'' are good for purifying plasmids, BL21(DE3) ''E. coli'' are more efficient for expressing protein. Thus, we transformed the plasmid into BL21(DE3) for the purposes of protein expression. We plated the bacteria on agar with the antibiotic ampicillin. While normal ''E. coli'' will die in the presence of ampicillin, the pET21-''yxiM'' plasmid has a gene that allows bacteria to become ampicillin resistant. Thus, only bacteria that were successfully transformed by the plasmid will survive on the ampicillin plate, allowing us to select for bacteria that have been transformed and thus bacteria that will express the protein YxiM.<br />
<br />
== Protein Expression ==<br />
<br />
After a day, colonies of transformed bacteria were visible on the agar plates. To express YxiM, we inoculated a single colony of bacteria into a liquid culture. In the plasmid, the ''yxiM'' gene is under control of the ''lac'' operon. This means that in the absence of an inducer, the transcription of the ''yxiM'' gene is repressed. Thus, we added IPTG, to activate the operon and drive the transcription of the protein YxiM.<br />
<br />
== Protein Purification ==<br />
<br />
After several hours, the bacteria have produced a relatively large amount of YxiM. To collect the protein, which at this point remained inside the cells, the bacteria were lysed (their cell walls were burst). The resulting mixture consisted of various cellular proteins and debris. The plasmid DNA sequence that coded for YxiM added a "tag" of histidines at the end of the protein. This allows us to separate YxiM proteins from the other types of proteins in the ''E. coli'' cells because when the mixture is passed through a nickel column, the tagged YxiM proteins stick to the column, while the other proteins flow through. Finally, we added elution buffer to the columns, which caused the proteins to detach from the nickel columns, creating a solution of just the YxiM proteins.<br />
<br />
== Esterase Activity Assay ==<br />
<br />
Now that we had purified protein, we could test the function of YxiM ''in vitro''. Since we believed that YxiM was an ester, we placed it in a buffered solution with 4-nitrophenyl butyrate, a type of ester. Esterases should hydrolyze 4-nitrophenyl butyrate, causing the products butyric acid and 4-nitrophenol to form. Since 4-nitrophenyl is a yellow color, the absorbance of the solution changes as more products are formed. We used colorimeter to measure the absorbance at 430 nm as a proxy for esterase activity. We found that the absorbance increases over time, which suggests that YxiM is indeed an esterase.<br />
<br />
Specifically, we found that the Lineweaver-Burk plot of esterase activity is linear. This is typical of enzymes, as predicted by the Michaelis-Menten model of enzyme kinetics. Technically, we did not construct a true Lineweaver-Burk plot, as we used absorbance as a proxy for molar concentration, but absorbance varies linearly with concentration, as shown by the Beer-Lambert law.<br />
<br />
== Discussion ==<br />
<br />
YxiM is a previously uncharacterized protein whose crystal structure has been solved and deposited in the PDB.<br />
<br />
Protein sequence analysis with BLAST shows that YxiM is likely an esterase. PyMOL shows that the top BLAST hits also align well with the 3D structure of the α-helix domain of YxiM. Almost all the top structural hits in Dali are esterases as well, and ProMOL shows that the active site of YxiM most resembles one of an esterase or protease. The same catalytic triad (S171, D339, H342) is implicated in both protease and esterase activity, suggesting YxiM could be a multifunctional hydrolase. The catalytic motif of the esterase 1BWR aligns particularly well with YxiM.<br />
<br />
We tested YxiM for esterase activity ''in vitro'' in an effort to confirm the ''in silico'' predictions. YxiM showed esterase activity on 4-nitrophenyl butyrate, as absorbance increased during the assay. The Lineweaver-Burk plot of YxiM esterase activity is linear, which is typical of enzymes.<br />
<br />
Thus, on the basis of protein sequence and structural analysis ''in silico'' and functional assays ''in vitro'', we conclude that YxiM is an esterase.<br />
<br />
== Future Directions ==<br />
<br />
To further confirm the activity of YxiM as an esterase, we can perform mutagenesis on the putative catalytic triad by performing PCR on the plasmid DNA with specialized primers. If we mutate the catalytic triad, then we expect that the protein will not be able to perform its function anymore. Through another round of transformation and purification of this mutated DNA, we would expect the protein to show no activity in our esterase assay.<br />
<br />
Our analysis suggests that YxiM could also demonstrate protease activity. To test this, we could perform protease assays as well.<br />
<br />
To further study enzyme kinetics, we could relate absorbance with concentration of protein. We can achieve this by performing a Bradford protein assay to compute the extinction coefficient.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=File:PET21-YxiM_Map.png&diff=2904767File:PET21-YxiM Map.png2018-05-25T10:12:16Z<p>Jennifer Taylor: </p>
<hr />
<div></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=File:YxiM_green-1J00_blue.png&diff=2904766File:YxiM green-1J00 blue.png2018-05-25T10:08:10Z<p>Jennifer Taylor: </p>
<hr />
<div></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=File:YxiM-1BWR_Catalytic.png&diff=2904765File:YxiM-1BWR Catalytic.png2018-05-25T10:07:56Z<p>Jennifer Taylor: uploaded a new version of "Image:YxiM-1BWR Catalytic.png"</p>
<hr />
<div></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=File:YxiM-1BWR_Catalytic.png&diff=2904764File:YxiM-1BWR Catalytic.png2018-05-25T10:03:42Z<p>Jennifer Taylor: </p>
<hr />
<div></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=File:YxiM_Lineweaver-Burk_Plot.png&diff=2904763File:YxiM Lineweaver-Burk Plot.png2018-05-25T10:01:29Z<p>Jennifer Taylor: </p>
<hr />
<div></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=File:YxiM_Purification.png&diff=2904762File:YxiM Purification.png2018-05-25T10:01:16Z<p>Jennifer Taylor: </p>
<hr />
<div></div>Jennifer Taylorhttps://proteopedia.org/index.php?title=File:YxiM_Expression.png&diff=2904761File:YxiM Expression.png2018-05-25T10:01:02Z<p>Jennifer Taylor: </p>
<hr />
<div></div>Jennifer Taylor