Thermal stability and kinetic constants for 129 variants of a family 1 glycoside hydrolase reveal that enzyme activity and stability can be separately designed
Accurate modeling of enzyme activity and stability is an important goal of the protein engineering community. However, studies seeking to evaluate current progress are limited by small data sets of quantitative kinetic constants and thermal stability measurements. Here, we report quantitative measur...
Gespeichert in:
| Veröffentlicht in: | PloS one 2017-05, Vol.12 (5), p.e0176255-e0176255 |
|---|---|
| Hauptverfasser: | , , , , , , , |
| Format: | Artikel |
| Sprache: | eng |
| Schlagworte: | |
| Online-Zugang: | Volltext |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| container_end_page | e0176255 |
|---|---|
| container_issue | 5 |
| container_start_page | e0176255 |
| container_title | PloS one |
| container_volume | 12 |
| creator | Carlin, Dylan Alexander Hapig-Ward, Siena Chan, Bill Wayne Damrau, Natalie Riley, Mary Caster, Ryan W Bethards, Bowen Siegel, Justin B |
| description | Accurate modeling of enzyme activity and stability is an important goal of the protein engineering community. However, studies seeking to evaluate current progress are limited by small data sets of quantitative kinetic constants and thermal stability measurements. Here, we report quantitative measurements of soluble protein expression in E. coli, thermal stability, and Michaelis-Menten constants (kcat, KM, and kcat/KM) for 129 designed mutants of a glycoside hydrolase. Statistical analyses reveal that functional Tm is independent of kcat, KM, and kcat/KM in this system, illustrating that an individual mutation can modulate these functional parameters independently. In addition, this data set is used to evaluate computational predictions of protein stability using the established Rosetta and FoldX algorithms. Predictions for both are found to correlate only weakly with experimental measurements, suggesting improvements are needed in the underlying algorithms. |
| doi_str_mv | 10.1371/journal.pone.0176255 |
| format | Article |
| fullrecord | <record><control><sourceid>gale_plos_</sourceid><recordid>TN_cdi_plos_journals_1901317083</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><galeid>A492500508</galeid><doaj_id>oai_doaj_org_article_37208aa02a7a40ffae4f3454e1a86f30</doaj_id><sourcerecordid>A492500508</sourcerecordid><originalsourceid>FETCH-LOGICAL-c692t-722fea61e6cbd84e5f0156bab6a5a22190e27c855f6375edc10b25d07fe916813</originalsourceid><addsrcrecordid>eNqNk9tu1DAQhiMEoqXwBggsISG42MWH2ElukKqKQ6VKlaBwa02S8a6LE29t74rlZXhVvN1t6aJeoFw4Gn_z__Z4piieMzplomLvLv0yjOCmCz_ilLJKcSkfFIesEXyiOBUP7_wfFE9ivKRUilqpx8UBr6VgrJaHxe-LOYYBHIkJWutsWhMYe_LDjphsRzo_5o0xRWJ8IIw3ZAXBXge8IUAMDNatCSMzt-58tD2S-boP3kFEEnCFWTnNIREcf60HJNAlu7ox-WvZwUhaJBEXECBhVuwx2tmI_dPikQEX8dluPSq-ffxwcfJ5cnb-6fTk-GzSqYanScW5QVAMVdf2dYnSUCZVC60CCZyzhiKvulpKo0Qlse8YbbnsaWWwYapm4qh4udVdOB_1rrZR50QmWEVrkYnTLdF7uNSLYAcIa-3B6uuADzMNIdfMoRYVpzUA5VBBSY0BLI0oZYkMamUEzVrvd27LdsinwTEFcHui-zujneuZX2lZikapKgu82QkEf7XEmPRgY4fOwYh-uT13JctKbrxe_YPef7sdNYN8ATsan327jag-Lhsuc-9k7KiY3kPlr8fB5l5BY3N8L-HtXkJmEv5MM1jGqE-_fvl_9vz7Pvv6DjvPbZbm0btlsrlh98FyC3bBxxjQ3BaZUb2Zo5tq6M0c6d0c5bQXdx_oNulmcMQf0sQZ-A</addsrcrecordid><sourcetype>Open Website</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>1901317083</pqid></control><display><type>article</type><title>Thermal stability and kinetic constants for 129 variants of a family 1 glycoside hydrolase reveal that enzyme activity and stability can be separately designed</title><source>MEDLINE</source><source>DOAJ Directory of Open Access Journals</source><source>Public Library of Science (PLoS) Journals Open Access</source><source>EZB-FREE-00999 freely available EZB journals</source><source>PubMed Central</source><source>Free Full-Text Journals in Chemistry</source><creator>Carlin, Dylan Alexander ; Hapig-Ward, Siena ; Chan, Bill Wayne ; Damrau, Natalie ; Riley, Mary ; Caster, Ryan W ; Bethards, Bowen ; Siegel, Justin B</creator><contributor>Soares, Claudio M</contributor><creatorcontrib>Carlin, Dylan Alexander ; Hapig-Ward, Siena ; Chan, Bill Wayne ; Damrau, Natalie ; Riley, Mary ; Caster, Ryan W ; Bethards, Bowen ; Siegel, Justin B ; Soares, Claudio M</creatorcontrib><description>Accurate modeling of enzyme activity and stability is an important goal of the protein engineering community. However, studies seeking to evaluate current progress are limited by small data sets of quantitative kinetic constants and thermal stability measurements. Here, we report quantitative measurements of soluble protein expression in E. coli, thermal stability, and Michaelis-Menten constants (kcat, KM, and kcat/KM) for 129 designed mutants of a glycoside hydrolase. Statistical analyses reveal that functional Tm is independent of kcat, KM, and kcat/KM in this system, illustrating that an individual mutation can modulate these functional parameters independently. In addition, this data set is used to evaluate computational predictions of protein stability using the established Rosetta and FoldX algorithms. Predictions for both are found to correlate only weakly with experimental measurements, suggesting improvements are needed in the underlying algorithms.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0176255</identifier><identifier>PMID: 28531185</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Algorithms ; Amino acid sequence ; Antibiotic resistance ; Barnase ; Benchmarks ; Biocatalysts ; Biochemistry ; Biology and Life Sciences ; Catalysis ; Catalysts ; Catalytic Domain ; Cloning, Molecular ; Computer applications ; Computer programs ; Crystal structure ; Crystallography, X-Ray ; Datasets ; Design ; Directed evolution ; E coli ; Electrostatic properties ; Engineering ; Enzymatic activity ; Enzyme activity ; Enzyme kinetics ; Enzyme Stability ; Enzymes ; Escherichia coli ; Escherichia coli - enzymology ; Escherichia coli - genetics ; Gene mutation ; Genetic Variation ; Genetics ; Genomes ; Glycosidases ; Glycoside hydrolase ; Glycoside Hydrolases - chemistry ; Glycoside Hydrolases - genetics ; Glycosides ; Hydrolases ; Hydrolysis ; Immunogenicity ; Influenza ; Inhibitors ; Kinetics ; Learning algorithms ; Machinery ; Macromolecules ; Models, Molecular ; Molecular modelling ; Mutagenesis ; Mutants ; Mutation ; Optimization ; Peptides ; Physical Sciences ; Protein expression ; Protein Structure, Tertiary ; Proteins ; Public access ; Research and Analysis Methods ; Residues ; Statistical analysis ; Temperature ; Thermal stability</subject><ispartof>PloS one, 2017-05, Vol.12 (5), p.e0176255-e0176255</ispartof><rights>COPYRIGHT 2017 Public Library of Science</rights><rights>2017 Carlin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2017 Carlin et al 2017 Carlin et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-722fea61e6cbd84e5f0156bab6a5a22190e27c855f6375edc10b25d07fe916813</citedby><cites>FETCH-LOGICAL-c692t-722fea61e6cbd84e5f0156bab6a5a22190e27c855f6375edc10b25d07fe916813</cites><orcidid>0000-0002-8729-734X</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5439667/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5439667/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,2102,2928,23866,27924,27925,53791,53793,79600,79601</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/28531185$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Soares, Claudio M</contributor><creatorcontrib>Carlin, Dylan Alexander</creatorcontrib><creatorcontrib>Hapig-Ward, Siena</creatorcontrib><creatorcontrib>Chan, Bill Wayne</creatorcontrib><creatorcontrib>Damrau, Natalie</creatorcontrib><creatorcontrib>Riley, Mary</creatorcontrib><creatorcontrib>Caster, Ryan W</creatorcontrib><creatorcontrib>Bethards, Bowen</creatorcontrib><creatorcontrib>Siegel, Justin B</creatorcontrib><title>Thermal stability and kinetic constants for 129 variants of a family 1 glycoside hydrolase reveal that enzyme activity and stability can be separately designed</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Accurate modeling of enzyme activity and stability is an important goal of the protein engineering community. However, studies seeking to evaluate current progress are limited by small data sets of quantitative kinetic constants and thermal stability measurements. Here, we report quantitative measurements of soluble protein expression in E. coli, thermal stability, and Michaelis-Menten constants (kcat, KM, and kcat/KM) for 129 designed mutants of a glycoside hydrolase. Statistical analyses reveal that functional Tm is independent of kcat, KM, and kcat/KM in this system, illustrating that an individual mutation can modulate these functional parameters independently. In addition, this data set is used to evaluate computational predictions of protein stability using the established Rosetta and FoldX algorithms. Predictions for both are found to correlate only weakly with experimental measurements, suggesting improvements are needed in the underlying algorithms.</description><subject>Algorithms</subject><subject>Amino acid sequence</subject><subject>Antibiotic resistance</subject><subject>Barnase</subject><subject>Benchmarks</subject><subject>Biocatalysts</subject><subject>Biochemistry</subject><subject>Biology and Life Sciences</subject><subject>Catalysis</subject><subject>Catalysts</subject><subject>Catalytic Domain</subject><subject>Cloning, Molecular</subject><subject>Computer applications</subject><subject>Computer programs</subject><subject>Crystal structure</subject><subject>Crystallography, X-Ray</subject><subject>Datasets</subject><subject>Design</subject><subject>Directed evolution</subject><subject>E coli</subject><subject>Electrostatic properties</subject><subject>Engineering</subject><subject>Enzymatic activity</subject><subject>Enzyme activity</subject><subject>Enzyme kinetics</subject><subject>Enzyme Stability</subject><subject>Enzymes</subject><subject>Escherichia coli</subject><subject>Escherichia coli - enzymology</subject><subject>Escherichia coli - genetics</subject><subject>Gene mutation</subject><subject>Genetic Variation</subject><subject>Genetics</subject><subject>Genomes</subject><subject>Glycosidases</subject><subject>Glycoside hydrolase</subject><subject>Glycoside Hydrolases - chemistry</subject><subject>Glycoside Hydrolases - genetics</subject><subject>Glycosides</subject><subject>Hydrolases</subject><subject>Hydrolysis</subject><subject>Immunogenicity</subject><subject>Influenza</subject><subject>Inhibitors</subject><subject>Kinetics</subject><subject>Learning algorithms</subject><subject>Machinery</subject><subject>Macromolecules</subject><subject>Models, Molecular</subject><subject>Molecular modelling</subject><subject>Mutagenesis</subject><subject>Mutants</subject><subject>Mutation</subject><subject>Optimization</subject><subject>Peptides</subject><subject>Physical Sciences</subject><subject>Protein expression</subject><subject>Protein Structure, Tertiary</subject><subject>Proteins</subject><subject>Public access</subject><subject>Research and Analysis Methods</subject><subject>Residues</subject><subject>Statistical analysis</subject><subject>Temperature</subject><subject>Thermal stability</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2017</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>DOA</sourceid><recordid>eNqNk9tu1DAQhiMEoqXwBggsISG42MWH2ElukKqKQ6VKlaBwa02S8a6LE29t74rlZXhVvN1t6aJeoFw4Gn_z__Z4piieMzplomLvLv0yjOCmCz_ilLJKcSkfFIesEXyiOBUP7_wfFE9ivKRUilqpx8UBr6VgrJaHxe-LOYYBHIkJWutsWhMYe_LDjphsRzo_5o0xRWJ8IIw3ZAXBXge8IUAMDNatCSMzt-58tD2S-boP3kFEEnCFWTnNIREcf60HJNAlu7ox-WvZwUhaJBEXECBhVuwx2tmI_dPikQEX8dluPSq-ffxwcfJ5cnb-6fTk-GzSqYanScW5QVAMVdf2dYnSUCZVC60CCZyzhiKvulpKo0Qlse8YbbnsaWWwYapm4qh4udVdOB_1rrZR50QmWEVrkYnTLdF7uNSLYAcIa-3B6uuADzMNIdfMoRYVpzUA5VBBSY0BLI0oZYkMamUEzVrvd27LdsinwTEFcHui-zujneuZX2lZikapKgu82QkEf7XEmPRgY4fOwYh-uT13JctKbrxe_YPef7sdNYN8ATsan327jag-Lhsuc-9k7KiY3kPlr8fB5l5BY3N8L-HtXkJmEv5MM1jGqE-_fvl_9vz7Pvv6DjvPbZbm0btlsrlh98FyC3bBxxjQ3BaZUb2Zo5tq6M0c6d0c5bQXdx_oNulmcMQf0sQZ-A</recordid><startdate>20170522</startdate><enddate>20170522</enddate><creator>Carlin, Dylan Alexander</creator><creator>Hapig-Ward, Siena</creator><creator>Chan, Bill Wayne</creator><creator>Damrau, Natalie</creator><creator>Riley, Mary</creator><creator>Caster, Ryan W</creator><creator>Bethards, Bowen</creator><creator>Siegel, Justin B</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope><orcidid>https://orcid.org/0000-0002-8729-734X</orcidid></search><sort><creationdate>20170522</creationdate><title>Thermal stability and kinetic constants for 129 variants of a family 1 glycoside hydrolase reveal that enzyme activity and stability can be separately designed</title><author>Carlin, Dylan Alexander ; Hapig-Ward, Siena ; Chan, Bill Wayne ; Damrau, Natalie ; Riley, Mary ; Caster, Ryan W ; Bethards, Bowen ; Siegel, Justin B</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c692t-722fea61e6cbd84e5f0156bab6a5a22190e27c855f6375edc10b25d07fe916813</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2017</creationdate><topic>Algorithms</topic><topic>Amino acid sequence</topic><topic>Antibiotic resistance</topic><topic>Barnase</topic><topic>Benchmarks</topic><topic>Biocatalysts</topic><topic>Biochemistry</topic><topic>Biology and Life Sciences</topic><topic>Catalysis</topic><topic>Catalysts</topic><topic>Catalytic Domain</topic><topic>Cloning, Molecular</topic><topic>Computer applications</topic><topic>Computer programs</topic><topic>Crystal structure</topic><topic>Crystallography, X-Ray</topic><topic>Datasets</topic><topic>Design</topic><topic>Directed evolution</topic><topic>E coli</topic><topic>Electrostatic properties</topic><topic>Engineering</topic><topic>Enzymatic activity</topic><topic>Enzyme activity</topic><topic>Enzyme kinetics</topic><topic>Enzyme Stability</topic><topic>Enzymes</topic><topic>Escherichia coli</topic><topic>Escherichia coli - enzymology</topic><topic>Escherichia coli - genetics</topic><topic>Gene mutation</topic><topic>Genetic Variation</topic><topic>Genetics</topic><topic>Genomes</topic><topic>Glycosidases</topic><topic>Glycoside hydrolase</topic><topic>Glycoside Hydrolases - chemistry</topic><topic>Glycoside Hydrolases - genetics</topic><topic>Glycosides</topic><topic>Hydrolases</topic><topic>Hydrolysis</topic><topic>Immunogenicity</topic><topic>Influenza</topic><topic>Inhibitors</topic><topic>Kinetics</topic><topic>Learning algorithms</topic><topic>Machinery</topic><topic>Macromolecules</topic><topic>Models, Molecular</topic><topic>Molecular modelling</topic><topic>Mutagenesis</topic><topic>Mutants</topic><topic>Mutation</topic><topic>Optimization</topic><topic>Peptides</topic><topic>Physical Sciences</topic><topic>Protein expression</topic><topic>Protein Structure, Tertiary</topic><topic>Proteins</topic><topic>Public access</topic><topic>Research and Analysis Methods</topic><topic>Residues</topic><topic>Statistical analysis</topic><topic>Temperature</topic><topic>Thermal stability</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Carlin, Dylan Alexander</creatorcontrib><creatorcontrib>Hapig-Ward, Siena</creatorcontrib><creatorcontrib>Chan, Bill Wayne</creatorcontrib><creatorcontrib>Damrau, Natalie</creatorcontrib><creatorcontrib>Riley, Mary</creatorcontrib><creatorcontrib>Caster, Ryan W</creatorcontrib><creatorcontrib>Bethards, Bowen</creatorcontrib><creatorcontrib>Siegel, Justin B</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Opposing Viewpoints</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Animal Behavior Abstracts</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Nursing & Allied Health Database</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Immunology Abstracts</collection><collection>Meteorological & Geoastrophysical Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Agricultural Science Collection</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Public Health Database</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Technology Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Materials Science & Engineering Collection</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>Advanced Technologies & Aerospace Collection</collection><collection>Agricultural & Environmental Science Collection</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Technology Collection</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Materials Science Collection</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Materials Science Database</collection><collection>Nursing & Allied Health Database (Alumni Edition)</collection><collection>Meteorological & Geoastrophysical Abstracts - Academic</collection><collection>ProQuest Engineering Collection</collection><collection>ProQuest Biological Science Collection</collection><collection>Agricultural Science Database</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Engineering Database</collection><collection>Nursing & Allied Health Premium</collection><collection>Advanced Technologies & Aerospace Database</collection><collection>ProQuest Advanced Technologies & Aerospace Collection</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Environmental Science Database</collection><collection>Materials Science Collection</collection><collection>Access via ProQuest (Open Access)</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>Engineering Collection</collection><collection>Environmental Science Collection</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Carlin, Dylan Alexander</au><au>Hapig-Ward, Siena</au><au>Chan, Bill Wayne</au><au>Damrau, Natalie</au><au>Riley, Mary</au><au>Caster, Ryan W</au><au>Bethards, Bowen</au><au>Siegel, Justin B</au><au>Soares, Claudio M</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Thermal stability and kinetic constants for 129 variants of a family 1 glycoside hydrolase reveal that enzyme activity and stability can be separately designed</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2017-05-22</date><risdate>2017</risdate><volume>12</volume><issue>5</issue><spage>e0176255</spage><epage>e0176255</epage><pages>e0176255-e0176255</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Accurate modeling of enzyme activity and stability is an important goal of the protein engineering community. However, studies seeking to evaluate current progress are limited by small data sets of quantitative kinetic constants and thermal stability measurements. Here, we report quantitative measurements of soluble protein expression in E. coli, thermal stability, and Michaelis-Menten constants (kcat, KM, and kcat/KM) for 129 designed mutants of a glycoside hydrolase. Statistical analyses reveal that functional Tm is independent of kcat, KM, and kcat/KM in this system, illustrating that an individual mutation can modulate these functional parameters independently. In addition, this data set is used to evaluate computational predictions of protein stability using the established Rosetta and FoldX algorithms. Predictions for both are found to correlate only weakly with experimental measurements, suggesting improvements are needed in the underlying algorithms.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>28531185</pmid><doi>10.1371/journal.pone.0176255</doi><tpages>e0176255</tpages><orcidid>https://orcid.org/0000-0002-8729-734X</orcidid><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2017-05, Vol.12 (5), p.e0176255-e0176255 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_1901317083 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Public Library of Science (PLoS) Journals Open Access; EZB-FREE-00999 freely available EZB journals; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Algorithms Amino acid sequence Antibiotic resistance Barnase Benchmarks Biocatalysts Biochemistry Biology and Life Sciences Catalysis Catalysts Catalytic Domain Cloning, Molecular Computer applications Computer programs Crystal structure Crystallography, X-Ray Datasets Design Directed evolution E coli Electrostatic properties Engineering Enzymatic activity Enzyme activity Enzyme kinetics Enzyme Stability Enzymes Escherichia coli Escherichia coli - enzymology Escherichia coli - genetics Gene mutation Genetic Variation Genetics Genomes Glycosidases Glycoside hydrolase Glycoside Hydrolases - chemistry Glycoside Hydrolases - genetics Glycosides Hydrolases Hydrolysis Immunogenicity Influenza Inhibitors Kinetics Learning algorithms Machinery Macromolecules Models, Molecular Molecular modelling Mutagenesis Mutants Mutation Optimization Peptides Physical Sciences Protein expression Protein Structure, Tertiary Proteins Public access Research and Analysis Methods Residues Statistical analysis Temperature Thermal stability |
| title | Thermal stability and kinetic constants for 129 variants of a family 1 glycoside hydrolase reveal that enzyme activity and stability can be separately designed |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-02T00%3A59%3A08IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Thermal%20stability%20and%20kinetic%20constants%20for%20129%20variants%20of%20a%20family%201%20glycoside%20hydrolase%20reveal%20that%20enzyme%20activity%20and%20stability%20can%20be%20separately%20designed&rft.jtitle=PloS%20one&rft.au=Carlin,%20Dylan%20Alexander&rft.date=2017-05-22&rft.volume=12&rft.issue=5&rft.spage=e0176255&rft.epage=e0176255&rft.pages=e0176255-e0176255&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0176255&rft_dat=%3Cgale_plos_%3EA492500508%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1901317083&rft_id=info:pmid/28531185&rft_galeid=A492500508&rft_doaj_id=oai_doaj_org_article_37208aa02a7a40ffae4f3454e1a86f30&rfr_iscdi=true |