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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Dynamic Requirements for a Functional Protein Hinge</title><author><name sortKey="Kempf, James G" sort="Kempf, James G" uniqKey="Kempf J" first="James G." last="Kempf">James G. Kempf</name><affiliation><nlm:aff id="A1">Department of Chemistry, Yale University, P.O. Box 208107, New Haven, CT 06520, USA</nlm:aff></affiliation></author><author><name sortKey="Jung, Ju Yeon" sort="Jung, Ju Yeon" uniqKey="Jung J" first="Ju-Yeon" last="Jung">Ju-Yeon Jung</name><affiliation><nlm:aff id="A2">Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, USA</nlm:aff></affiliation></author><author><name sortKey="Ragain, Christina" sort="Ragain, Christina" uniqKey="Ragain C" first="Christina" last="Ragain">Christina Ragain</name><affiliation><nlm:aff id="A1">Department of Chemistry, Yale University, P.O. Box 208107, New Haven, CT 06520, USA</nlm:aff></affiliation></author><author><name sortKey="Sampson, Nicole S" sort="Sampson, Nicole S" uniqKey="Sampson N" first="Nicole S." last="Sampson">Nicole S. Sampson</name><affiliation><nlm:aff id="A2">Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, USA</nlm:aff></affiliation></author><author><name sortKey="Loria, J Patrick" sort="Loria, J Patrick" uniqKey="Loria J" first="J. Patrick" last="Loria">J. Patrick Loria</name><affiliation><nlm:aff id="A1">Department of Chemistry, Yale University, P.O. Box 208107, New Haven, CT 06520, USA</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">17336327</idno><idno type="pmc">2203303</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2203303</idno><idno type="RBID">PMC:2203303</idno><idno type="doi">10.1016/j.jmb.2007.01.074</idno><date when="2007">2007</date><idno type="wicri:Area/Pmc/Corpus">000016</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000016</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Dynamic Requirements for a Functional Protein Hinge</title><author><name sortKey="Kempf, James G" sort="Kempf, James G" uniqKey="Kempf J" first="James G." last="Kempf">James G. Kempf</name><affiliation><nlm:aff id="A1">Department of Chemistry, Yale University, P.O. Box 208107, New Haven, CT 06520, USA</nlm:aff></affiliation></author><author><name sortKey="Jung, Ju Yeon" sort="Jung, Ju Yeon" uniqKey="Jung J" first="Ju-Yeon" last="Jung">Ju-Yeon Jung</name><affiliation><nlm:aff id="A2">Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, USA</nlm:aff></affiliation></author><author><name sortKey="Ragain, Christina" sort="Ragain, Christina" uniqKey="Ragain C" first="Christina" last="Ragain">Christina Ragain</name><affiliation><nlm:aff id="A1">Department of Chemistry, Yale University, P.O. Box 208107, New Haven, CT 06520, USA</nlm:aff></affiliation></author><author><name sortKey="Sampson, Nicole S" sort="Sampson, Nicole S" uniqKey="Sampson N" first="Nicole S." last="Sampson">Nicole S. Sampson</name><affiliation><nlm:aff id="A2">Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, USA</nlm:aff></affiliation></author><author><name sortKey="Loria, J Patrick" sort="Loria, J Patrick" uniqKey="Loria J" first="J. Patrick" last="Loria">J. Patrick Loria</name><affiliation><nlm:aff id="A1">Department of Chemistry, Yale University, P.O. Box 208107, New Haven, CT 06520, USA</nlm:aff></affiliation></author></analytic><series><title level="j">Journal of molecular biology</title><idno type="ISSN">0022-2836</idno><idno type="eISSN">1089-8638</idno><imprint><date when="2007">2007</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><title>Summary</title><p id="P3">The enzyme triosephosphate isomerase (TIM) is a model of catalytic efficiency. The 11-residue loop 6 at the TIM active site plays a major role in this enzymatic prowess. The loop moves between open and closed states, which facilitate substrate access and catalysis, respectively. The N- and C-terminal hinges of loop 6 control this motion. Here, we detail flexibility requirements for hinges in a comparative solution NMR study of wild-type (WT) TIM and a quintuple mutant (PGG/GGG). The latter contained glycine substitutions in the N-terminal hinge at Val167 and Trp168, which follow the essential Pro166, and in the C-terminal hinge at Lys174, Thr175, and Ala176. Previous work demonstrated that PGG/GGG has a 10-fold higher K<sub>m</sub> value and 10<sup>3</sup>-fold reduced <italic>k</italic><sub>cat</sub> relative to WT with either [D]-glyceraldehyde 3-phosphate or dihyrdroxyacetone phosphate as substrate. Our NMR results explain this in terms of altered loop-6 dynamics in PGG/GGG. In the mutant, loop 6 exhibits conformational heterogeneity with corresponding motional rates < 750 s<sup>−1</sup> that are an order of magnitude slower than the natural WT loop-6 motion. At the same time, ns-timescale motions of loop 6 are greatly enhanced in the mutant relative to WT. These differences from WT behavior occur in both apo PGG/GGG and in the form bound to the reaction-intermediate analog, 2-phosphoglycolate (2-PGA). In addition, as indicated by <sup>1</sup>H, <sup>15</sup>N and <sup>13</sup>CO chemical-shifts, the glycine substitutions (a) diminished the enzyme’s response to ligand, and (b) induced structural perturbations in apo and 2-PGA-bound forms of TIM that are atypical of those observed in WT. Altogether, these data show that PGG/GGG exists in multiple conformations that are not fully competent for ligand binding or catalysis. These experiments elucidate an important principle of catalytic hinge design in proteins: structural rigidity is essential for focused motional freedom of active-site loops.</p></div></front></TEI><pmc article-type="research-article"><pmc-comment>The publisher of this article does not allow downloading of the full text in XML form.</pmc-comment>
<pmc-dir>properties manuscript</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-journal-id">2985088R</journal-id><journal-id journal-id-type="pubmed-jr-id">4967</journal-id><journal-id journal-id-type="nlm-ta">J Mol Biol</journal-id><journal-title>Journal of molecular biology</journal-title><issn pub-type="ppub">0022-2836</issn><issn pub-type="epub">1089-8638</issn></journal-meta><article-meta><article-id pub-id-type="pmid">17336327</article-id><article-id pub-id-type="pmc">2203303</article-id><article-id pub-id-type="doi">10.1016/j.jmb.2007.01.074</article-id><article-id pub-id-type="manuscript">NIHMS21086</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Dynamic Requirements for a Functional Protein Hinge</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Kempf</surname><given-names>James G.</given-names></name><xref rid="A1" ref-type="aff">1</xref><xref rid="FN2" ref-type="author-notes">3</xref></contrib><contrib contrib-type="author"><name><surname>Jung</surname><given-names>Ju-yeon</given-names></name><xref rid="A2" ref-type="aff">2</xref><xref rid="FN3" ref-type="author-notes">4</xref></contrib><contrib contrib-type="author"><name><surname>Ragain</surname><given-names>Christina</given-names></name><xref rid="A1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Sampson</surname><given-names>Nicole S.</given-names></name><xref rid="A2" ref-type="aff">2</xref></contrib><contrib contrib-type="author"><name><surname>Loria</surname><given-names>J. Patrick</given-names></name><xref rid="A1" ref-type="aff">1</xref><xref rid="FN1" ref-type="author-notes">*</xref></contrib></contrib-group><aff id="A1"><label>1</label>Department of Chemistry, Yale University, P.O. Box 208107, New Haven, CT 06520, USA</aff><aff id="A2"><label>2</label>Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, USA</aff><author-notes><corresp id="FN1">*Corresponding Author: email: <email>patrick.loria@yale.edu</email>; phone: (203) 436-4847; fax: (203) 432-6144</corresp><fn id="FN2" fn-type="present-address"><label>3</label><p>Current address: Department of Chemistry, Rensselaer Polytechnic Institute, Troy, New York 12180, USA;</p></fn><fn id="FN3" fn-type="present-address"><label>4</label><p>Current address: Department of Genetic Medicine, Weill Medical College of Cornell University, New York, New York 10021</p></fn></author-notes><pub-date pub-type="nihms-submitted"><day>9</day><month>4</month><year>2007</year></pub-date><pub-date pub-type="epub"><day>3</day><month>2</month><year>2007</year></pub-date><pub-date pub-type="ppub"><day>20</day><month>4</month><year>2007</year></pub-date><pub-date pub-type="pmc-release"><day>20</day><month>4</month><year>2008</year></pub-date><volume>368</volume><issue>1</issue><fpage>131</fpage><lpage>149</lpage><abstract><title>Summary</title><p id="P3">The enzyme triosephosphate isomerase (TIM) is a model of catalytic efficiency. The 11-residue loop 6 at the TIM active site plays a major role in this enzymatic prowess. The loop moves between open and closed states, which facilitate substrate access and catalysis, respectively. The N- and C-terminal hinges of loop 6 control this motion. Here, we detail flexibility requirements for hinges in a comparative solution NMR study of wild-type (WT) TIM and a quintuple mutant (PGG/GGG). The latter contained glycine substitutions in the N-terminal hinge at Val167 and Trp168, which follow the essential Pro166, and in the C-terminal hinge at Lys174, Thr175, and Ala176. Previous work demonstrated that PGG/GGG has a 10-fold higher K<sub>m</sub> value and 10<sup>3</sup>-fold reduced <italic>k</italic><sub>cat</sub> relative to WT with either [D]-glyceraldehyde 3-phosphate or dihyrdroxyacetone phosphate as substrate. Our NMR results explain this in terms of altered loop-6 dynamics in PGG/GGG. In the mutant, loop 6 exhibits conformational heterogeneity with corresponding motional rates < 750 s<sup>−1</sup> that are an order of magnitude slower than the natural WT loop-6 motion. At the same time, ns-timescale motions of loop 6 are greatly enhanced in the mutant relative to WT. These differences from WT behavior occur in both apo PGG/GGG and in the form bound to the reaction-intermediate analog, 2-phosphoglycolate (2-PGA). In addition, as indicated by <sup>1</sup>H, <sup>15</sup>N and <sup>13</sup>CO chemical-shifts, the glycine substitutions (a) diminished the enzyme’s response to ligand, and (b) induced structural perturbations in apo and 2-PGA-bound forms of TIM that are atypical of those observed in WT. Altogether, these data show that PGG/GGG exists in multiple conformations that are not fully competent for ligand binding or catalysis. These experiments elucidate an important principle of catalytic hinge design in proteins: structural rigidity is essential for focused motional freedom of active-site loops.</p></abstract><kwd-group><kwd>Enzyme catalysis</kwd><kwd>Loop motion</kwd><kwd>Protein hinge</kwd><kwd>NMR spectroscopy</kwd><kwd>Protein dynamics</kwd><kwd>Triosephosphate isomerase</kwd></kwd-group><contract-num rid="GM1">R01 GM070823-01A1</contract-num><contract-sponsor id="GM1">National Institute of General Medical Sciences : NIGMS</contract-sponsor></article-meta></front></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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