microplate-based evaluation of complex denaturation pathways: Structural stability of Escherichia coli transketolase
We have previously developed a rapid microplate-based approach for measuring the denaturation curves by intrinsic tryptophan fluorescence for simple monomeric and two-state unfolding proteins. Here we demonstrate that it can accurately resolve the multiple conformational transitions that occur durin...
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| Veröffentlicht in: | Biotechnology and bioengineering 2008-04, Vol.99 (6), p.1303-1310 |
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| creator | Aucamp, Jean P Martinez-Torres, Ruben J Hibbert, Edward G Dalby, Paul A |
| description | We have previously developed a rapid microplate-based approach for measuring the denaturation curves by intrinsic tryptophan fluorescence for simple monomeric and two-state unfolding proteins. Here we demonstrate that it can accurately resolve the multiple conformational transitions that occur during the denaturation of a complex multimeric and cofactor associated protein. We have also analyzed the effect of two active-site mutations, D381A and Y440A upon the denaturation pathway of transketolase using intrinsic fluorescence measurements, and we compare the results from classical and microplate-based instrumentation. This work shows that the rapid assay is able to identify changes in the denaturation pathway, due to mutations or removal of cofactors, which affect the stability of the native and intermediate states. This would be of significant benefit for the directed evolution of protein stability, optimizing enzyme stability under biocatalytic process conditions, and also for engineering specific transitions in protein unfolding pathways. Biotechnol. Bioeng. 2008;99: 1303-1310. |
| doi_str_mv | 10.1002/bit.21705 |
| format | Article |
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Here we demonstrate that it can accurately resolve the multiple conformational transitions that occur during the denaturation of a complex multimeric and cofactor associated protein. We have also analyzed the effect of two active-site mutations, D381A and Y440A upon the denaturation pathway of transketolase using intrinsic fluorescence measurements, and we compare the results from classical and microplate-based instrumentation. This work shows that the rapid assay is able to identify changes in the denaturation pathway, due to mutations or removal of cofactors, which affect the stability of the native and intermediate states. This would be of significant benefit for the directed evolution of protein stability, optimizing enzyme stability under biocatalytic process conditions, and also for engineering specific transitions in protein unfolding pathways. Biotechnol. Bioeng. 2008;99: 1303-1310.</description><identifier>ISSN: 0006-3592</identifier><identifier>EISSN: 1097-0290</identifier><identifier>DOI: 10.1002/bit.21705</identifier><identifier>PMID: 17969139</identifier><identifier>CODEN: BIBIAU</identifier><language>eng</language><publisher>Hoboken: Wiley Subscription Services, Inc., A Wiley Company</publisher><subject>biocatalysis ; Biochemistry ; Catalysis ; denaturation ; directed evolution ; E coli ; Effects ; Enzyme Stability ; Escherichia coli ; Escherichia coli - enzymology ; high-throughput screening ; Mutation ; Protein Array Analysis - methods ; Protein Denaturation ; Protein folding ; Signal Transduction - physiology ; thermostability ; Transitions ; transketolase ; Transketolase - chemistry ; Transketolase - metabolism ; tryptophan fluorescence</subject><ispartof>Biotechnology and bioengineering, 2008-04, Vol.99 (6), p.1303-1310</ispartof><rights>Copyright © 2007 Wiley Periodicals, Inc.</rights><rights>Copyright 2007 Wiley Periodicals, Inc.</rights><rights>Copyright John Wiley and Sons, Limited Apr 15, 2008</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4745-9fb8407adf460be031444d9b8910a5d3908a4ad2fed8f6f3115169a26ac64f363</citedby><cites>FETCH-LOGICAL-c4745-9fb8407adf460be031444d9b8910a5d3908a4ad2fed8f6f3115169a26ac64f363</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1002%2Fbit.21705$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Fbit.21705$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,776,780,1411,27903,27904,45553,45554</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/17969139$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Aucamp, Jean P</creatorcontrib><creatorcontrib>Martinez-Torres, Ruben J</creatorcontrib><creatorcontrib>Hibbert, Edward G</creatorcontrib><creatorcontrib>Dalby, Paul A</creatorcontrib><title>microplate-based evaluation of complex denaturation pathways: Structural stability of Escherichia coli transketolase</title><title>Biotechnology and bioengineering</title><addtitle>Biotechnol. Bioeng</addtitle><description>We have previously developed a rapid microplate-based approach for measuring the denaturation curves by intrinsic tryptophan fluorescence for simple monomeric and two-state unfolding proteins. Here we demonstrate that it can accurately resolve the multiple conformational transitions that occur during the denaturation of a complex multimeric and cofactor associated protein. We have also analyzed the effect of two active-site mutations, D381A and Y440A upon the denaturation pathway of transketolase using intrinsic fluorescence measurements, and we compare the results from classical and microplate-based instrumentation. This work shows that the rapid assay is able to identify changes in the denaturation pathway, due to mutations or removal of cofactors, which affect the stability of the native and intermediate states. This would be of significant benefit for the directed evolution of protein stability, optimizing enzyme stability under biocatalytic process conditions, and also for engineering specific transitions in protein unfolding pathways. Biotechnol. Bioeng. 2008;99: 1303-1310.</description><subject>biocatalysis</subject><subject>Biochemistry</subject><subject>Catalysis</subject><subject>denaturation</subject><subject>directed evolution</subject><subject>E coli</subject><subject>Effects</subject><subject>Enzyme Stability</subject><subject>Escherichia coli</subject><subject>Escherichia coli - enzymology</subject><subject>high-throughput screening</subject><subject>Mutation</subject><subject>Protein Array Analysis - methods</subject><subject>Protein Denaturation</subject><subject>Protein folding</subject><subject>Signal Transduction - physiology</subject><subject>thermostability</subject><subject>Transitions</subject><subject>transketolase</subject><subject>Transketolase - chemistry</subject><subject>Transketolase - metabolism</subject><subject>tryptophan fluorescence</subject><issn>0006-3592</issn><issn>1097-0290</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2008</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqF0V9r1TAYBvAiijubXvgFtHgheNHtzZ8mjXc65nEwVNiG4k1426aebGl7TNJt59ubY48KguwqJPzeJ7w8WfaMwCEBoEe1jYeUSCgfZAsCShZAFTzMFgAgClYqupfth3CVrrIS4nG2R6QSijC1yGJvGz-uHUZT1BhMm5sbdBNGOw752OXN2K-ductbM2Cc_Py-xri6xU14k59HPzXbd5eHiLV1Nm62YyehWRlvm5XFFOFsHj0O4drE0aVPnmSPOnTBPN2dB9nl-5OL4w_F2afl6fHbs6LhkpeF6uqKg8S24wJqA4xwzltVV4oAli1TUCHHlnamrTrRMUJKIhRSgY3gHRPsIHs15679-GMyIerehsY4h4MZp6AlJMQZ3AsZ5VQofj8kSpRQiTLBl__Aq3HyQ9pWU8KkoKmthF7PKFUQgjedXnvbo99oAnrbrE7N6l_NJvt8FzjVvWn_yl2VCRzN4NY6s_l_kn53evE7spgnbIjm7s8E-mstJJOl_vJxqcVX9ll8U0tdJf9i9h2OGr97G_TlOQXCIK1cVUDZTyXDxms</recordid><startdate>20080415</startdate><enddate>20080415</enddate><creator>Aucamp, Jean P</creator><creator>Martinez-Torres, Ruben J</creator><creator>Hibbert, Edward G</creator><creator>Dalby, Paul A</creator><general>Wiley Subscription Services, Inc., A Wiley Company</general><general>Wiley Subscription Services, Inc</general><scope>FBQ</scope><scope>BSCLL</scope><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>7QF</scope><scope>7QO</scope><scope>7QQ</scope><scope>7SC</scope><scope>7SE</scope><scope>7SP</scope><scope>7SR</scope><scope>7T7</scope><scope>7TA</scope><scope>7TB</scope><scope>7U5</scope><scope>8BQ</scope><scope>8FD</scope><scope>C1K</scope><scope>F28</scope><scope>FR3</scope><scope>H8D</scope><scope>H8G</scope><scope>JG9</scope><scope>JQ2</scope><scope>KR7</scope><scope>L7M</scope><scope>L~C</scope><scope>L~D</scope><scope>P64</scope><scope>7QL</scope><scope>7X8</scope></search><sort><creationdate>20080415</creationdate><title>microplate-based evaluation of complex denaturation pathways: Structural stability of Escherichia coli transketolase</title><author>Aucamp, Jean P ; 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| ispartof | Biotechnology and bioengineering, 2008-04, Vol.99 (6), p.1303-1310 |
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| subjects | biocatalysis Biochemistry Catalysis denaturation directed evolution E coli Effects Enzyme Stability Escherichia coli Escherichia coli - enzymology high-throughput screening Mutation Protein Array Analysis - methods Protein Denaturation Protein folding Signal Transduction - physiology thermostability Transitions transketolase Transketolase - chemistry Transketolase - metabolism tryptophan fluorescence |
| title | microplate-based evaluation of complex denaturation pathways: Structural stability of Escherichia coli transketolase |
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