The Flexibility of a Distant Loop Modulates Active-Site Motion and Product Release in Ribonuclease A
Identifieur interne : 003792 ( Main/Exploration ); précédent : 003791; suivant : 003793The Flexibility of a Distant Loop Modulates Active-Site Motion and Product Release in Ribonuclease A
Auteurs : Nicolas Doucet [États-Unis] ; Eric D. Watt [États-Unis] ; J. Patrick Loria [États-Unis]Source :
- Biochemistry [ 0006-2960 ] ; 2009.
Abstract
The role of the flexible loop 1 in protein conformational motion and the dissociation of enzymatic product from Ribonuclease A (RNase A) was investigated by creation of a chimeric enzyme in which a six residue loop 1 from the RNase A homolog, eosinophil cationic protein (ECP) replaced the twelve residue loop in RNase A. The chimera (RNase AECP) experiences only local perturbations in NMR backbone chemical shifts compared to WT RNase A. Many of the flexible residues that were previously identified in WT as involved in an important conformational change now experience no NMR-detected millisecond motions in the chimera. Likewise, binding of the product analog, 3′-CMP to RNase AECP results in only minor chemical shift changes in the enzyme similar to what is observed for the H48A mutant of RNase A and in contrast to WT enzyme. For both RNase AECP and H48A there is a 10-fold decrease in the product release rate constant, koff compared to WT and in agreement with previous studies indicating the importance of flexibility in RNase A in the overall rate-limiting product release step. Together these NMR and biochemical experiments provide additional insight into the mechanism of millisecond motions in the RNase A catalytic cycle.
Url:
DOI: 10.1021/bi900830g
PubMed: 19588901
PubMed Central: 2741010
Affiliations:
Links toward previous steps (curation, corpus...)
- to stream Pmc, to step Corpus: 000042
- to stream Pmc, to step Curation: 000042
- to stream Pmc, to step Checkpoint: 000075
- to stream Ncbi, to step Merge: 000084
- to stream Ncbi, to step Curation: 000082
- to stream Ncbi, to step Checkpoint: 000082
- to stream Main, to step Merge: 003869
- to stream Main, to step Curation: 003792
Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">The Flexibility of a Distant Loop Modulates Active-Site Motion and Product Release in Ribonuclease A</title><author><name sortKey="Doucet, Nicolas" sort="Doucet, Nicolas" uniqKey="Doucet N" first="Nicolas" last="Doucet">Nicolas Doucet</name><affiliation wicri:level="2"><nlm:aff id="A1"> Department of Chemistry, Yale University, New Haven, Connecticut 06520</nlm:aff><country xml:lang="fr">États-Unis</country><placeName><region type="state">Connecticut</region></placeName><wicri:cityArea> Department of Chemistry, Yale University, New Haven</wicri:cityArea></affiliation></author><author><name sortKey="Watt, Eric D" sort="Watt, Eric D" uniqKey="Watt E" first="Eric D." last="Watt">Eric D. Watt</name><affiliation wicri:level="2"><nlm:aff id="A1"> Department of Chemistry, Yale University, New Haven, Connecticut 06520</nlm:aff><country xml:lang="fr">États-Unis</country><placeName><region type="state">Connecticut</region></placeName><wicri:cityArea> Department of Chemistry, Yale University, New Haven</wicri:cityArea></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 wicri:level="2"><nlm:aff id="A1"> Department of Chemistry, Yale University, New Haven, Connecticut 06520</nlm:aff><country xml:lang="fr">États-Unis</country><placeName><region type="state">Connecticut</region></placeName><wicri:cityArea> Department of Chemistry, Yale University, New Haven</wicri:cityArea></affiliation><affiliation wicri:level="2"><nlm:aff id="A2"> Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520</nlm:aff><country xml:lang="fr">États-Unis</country><placeName><region type="state">Connecticut</region></placeName><wicri:cityArea> Department of Molecular Biophysics and Biochemistry, Yale University, New Haven</wicri:cityArea></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">19588901</idno><idno type="pmc">2741010</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2741010</idno><idno type="RBID">PMC:2741010</idno><idno type="doi">10.1021/bi900830g</idno><date when="2009">2009</date><idno type="wicri:Area/Pmc/Corpus">000042</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000042</idno><idno type="wicri:Area/Pmc/Curation">000042</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000042</idno><idno type="wicri:Area/Pmc/Checkpoint">000075</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Checkpoint">000075</idno><idno type="wicri:Area/Ncbi/Merge">000084</idno><idno type="wicri:Area/Ncbi/Curation">000082</idno><idno type="wicri:Area/Ncbi/Checkpoint">000082</idno><idno type="wicri:doubleKey">0006-2960:2009:Doucet N:the:flexibility:of</idno><idno type="wicri:Area/Main/Merge">003869</idno><idno type="wicri:Area/Main/Curation">003792</idno><idno type="wicri:Area/Main/Exploration">003792</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">The Flexibility of a Distant Loop Modulates Active-Site Motion and Product Release in Ribonuclease A</title><author><name sortKey="Doucet, Nicolas" sort="Doucet, Nicolas" uniqKey="Doucet N" first="Nicolas" last="Doucet">Nicolas Doucet</name><affiliation wicri:level="2"><nlm:aff id="A1"> Department of Chemistry, Yale University, New Haven, Connecticut 06520</nlm:aff><country xml:lang="fr">États-Unis</country><placeName><region type="state">Connecticut</region></placeName><wicri:cityArea> Department of Chemistry, Yale University, New Haven</wicri:cityArea></affiliation></author><author><name sortKey="Watt, Eric D" sort="Watt, Eric D" uniqKey="Watt E" first="Eric D." last="Watt">Eric D. Watt</name><affiliation wicri:level="2"><nlm:aff id="A1"> Department of Chemistry, Yale University, New Haven, Connecticut 06520</nlm:aff><country xml:lang="fr">États-Unis</country><placeName><region type="state">Connecticut</region></placeName><wicri:cityArea> Department of Chemistry, Yale University, New Haven</wicri:cityArea></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 wicri:level="2"><nlm:aff id="A1"> Department of Chemistry, Yale University, New Haven, Connecticut 06520</nlm:aff><country xml:lang="fr">États-Unis</country><placeName><region type="state">Connecticut</region></placeName><wicri:cityArea> Department of Chemistry, Yale University, New Haven</wicri:cityArea></affiliation><affiliation wicri:level="2"><nlm:aff id="A2"> Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520</nlm:aff><country xml:lang="fr">États-Unis</country><placeName><region type="state">Connecticut</region></placeName><wicri:cityArea> Department of Molecular Biophysics and Biochemistry, Yale University, New Haven</wicri:cityArea></affiliation></author></analytic><series><title level="j">Biochemistry</title><idno type="ISSN">0006-2960</idno><idno type="eISSN">1520-4995</idno><imprint><date when="2009">2009</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p id="P1">The role of the flexible loop 1 in protein conformational motion and the dissociation of enzymatic product from Ribonuclease A (RNase A) was investigated by creation of a chimeric enzyme in which a six residue loop 1 from the RNase A homolog, eosinophil cationic protein (ECP) replaced the twelve residue loop in RNase A. The chimera (RNase A<sub>ECP</sub>) experiences only local perturbations in NMR backbone chemical shifts compared to WT RNase A. Many of the flexible residues that were previously identified in WT as involved in an important conformational change now experience no NMR-detected millisecond motions in the chimera. Likewise, binding of the product analog, 3′-CMP to RNase A<sub>ECP</sub> results in only minor chemical shift changes in the enzyme similar to what is observed for the H48A mutant of RNase A and in contrast to WT enzyme. For both RNase A<sub>ECP</sub> and H48A there is a 10-fold decrease in the product release rate constant, k<sub>off</sub> compared to WT and in agreement with previous studies indicating the importance of flexibility in RNase A in the overall rate-limiting product release step. Together these NMR and biochemical experiments provide additional insight into the mechanism of millisecond motions in the RNase A catalytic cycle.</p></div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>Connecticut</li></region></list><tree><country name="États-Unis"><region name="Connecticut"><name sortKey="Doucet, Nicolas" sort="Doucet, Nicolas" uniqKey="Doucet N" first="Nicolas" last="Doucet">Nicolas Doucet</name></region><name sortKey="Loria, J Patrick" sort="Loria, J Patrick" uniqKey="Loria J" first="J. Patrick" last="Loria">J. Patrick Loria</name><name sortKey="Loria, J Patrick" sort="Loria, J Patrick" uniqKey="Loria J" first="J. Patrick" last="Loria">J. Patrick Loria</name><name sortKey="Watt, Eric D" sort="Watt, Eric D" uniqKey="Watt E" first="Eric D." last="Watt">Eric D. Watt</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Wicri/Lorraine/explor/InforLorV4/Data/Main/Exploration
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 003792 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Main/Exploration/biblio.hfd -nk 003792 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Wicri/Lorraine
|area= InforLorV4
|flux= Main
|étape= Exploration
|type= RBID
|clé= PMC:2741010
|texte= The Flexibility of a Distant Loop Modulates Active-Site Motion and Product Release in Ribonuclease A
}}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/Main/Exploration/RBID.i -Sk "pubmed:19588901" \
| HfdSelect -Kh $EXPLOR_AREA/Data/Main/Exploration/biblio.hfd \
| NlmPubMed2Wicri -a InforLorV4
| This area was generated with Dilib version V0.6.33. |  |