Mechanistic insight provided by glutaredoxin within a fusion to redox-sensitive yellow fluorescent protein.
Identifieur interne : 000D36 ( Main/Exploration ); précédent : 000D35; suivant : 000D37Mechanistic insight provided by glutaredoxin within a fusion to redox-sensitive yellow fluorescent protein.
Auteurs : Olof Björnberg [Danemark] ; Henrik Stergaard ; Jakob R. WintherSource :
- Biochemistry [ 0006-2960 ] ; 2006.
Descripteurs français
- KwdFr :
- Cystamine (composition chimique), Disulfure de glutathion (métabolisme), Disulfures (composition chimique), Glutarédoxines (MeSH), Oxidoreductases (génétique), Oxidoreductases (métabolisme), Oxydoréduction (MeSH), Peroxyde d'hydrogène (composition chimique), Protéines bactériennes (métabolisme), Protéines de fusion recombinantes (métabolisme), Protéines luminescentes (métabolisme), Éthanol (analogues et dérivés), Éthanol (composition chimique).
- MESH :
- analogues et dérivés : Éthanol.
- composition chimique : Cystamine, Disulfures, Peroxyde d'hydrogène, Éthanol.
- génétique : Oxidoreductases.
- métabolisme : Disulfure de glutathion, Oxidoreductases, Protéines bactériennes, Protéines de fusion recombinantes, Protéines luminescentes.
- Glutarédoxines, Oxydoréduction.
English descriptors
- KwdEn :
- Bacterial Proteins (metabolism), Cystamine (chemistry), Disulfides (chemistry), Ethanol (analogs & derivatives), Ethanol (chemistry), Glutaredoxins (MeSH), Glutathione Disulfide (metabolism), Hydrogen Peroxide (chemistry), Luminescent Proteins (metabolism), Oxidation-Reduction (MeSH), Oxidoreductases (genetics), Oxidoreductases (metabolism), Recombinant Fusion Proteins (metabolism).
- MESH :
- chemical , analogs & derivatives : Ethanol.
- chemical , chemistry : Cystamine, Disulfides, Ethanol, Hydrogen Peroxide.
- chemical , genetics : Oxidoreductases.
- chemical , metabolism : Bacterial Proteins, Glutathione Disulfide, Luminescent Proteins, Oxidoreductases, Recombinant Fusion Proteins.
- chemical : Glutaredoxins.
- Oxidation-Reduction.
Abstract
Redox-sensitive yellow fluorescent protein (rxYFP) contains a dithiol disulfide pair that is thermodynamically suitable for monitoring intracellular glutathione redox potential. Glutaredoxin 1 (Grx1p) from yeast is known to catalyze the redox equilibrium between rxYFP and glutathione, and here, we have generated a fusion of the two proteins, rxYFP-Grx1p. In comparison to isolated subunits, intramolecular transfer of reducing equivalents made the fusion protein kinetically superior in reactions with glutathione. The rate of GSSG oxidation was thus improved by a factor of 3300. The reaction with GSSG most likely takes place entirely through a glutathionylated intermediate and not through transfer of an intramolecular disulfide bond. However, during oxidation by H(2)O(2), hydroxyethyl disulfide, or cystine, the glutaredoxin domain reacted first, followed by a rate-limiting (0.13 min(-)(1)) transfer of a disulfide bond to the other domain. Thus, reactivity toward other oxidants remains low, giving almost absolute glutathione specificity. We have further studied CPYC --> CPYS variants in the active site of Grx1p and found that the single Cys variant had elevated oxidoreductase activity separately and in the fusion. This could not be ascribed to the lack of an unproductive side reaction to glutaredoxin disulfide. Instead, slower alkylation kinetics with iodoacetamide indicates a better leaving-group capability of the remaining cysteine residue, which can explain the increased activity.
DOI: 10.1021/bi0522495
PubMed: 16475825
Affiliations:
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Winther</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2006">2006</date><idno type="RBID">pubmed:16475825</idno><idno type="pmid">16475825</idno><idno type="doi">10.1021/bi0522495</idno><idno type="wicri:Area/Main/Corpus">000D65</idno><idno type="wicri:explorRef" wicri:stream="Main" wicri:step="Corpus" wicri:corpus="PubMed">000D65</idno><idno type="wicri:Area/Main/Curation">000D65</idno><idno type="wicri:explorRef" wicri:stream="Main" wicri:step="Curation">000D65</idno><idno type="wicri:Area/Main/Exploration">000D65</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Mechanistic insight provided by glutaredoxin within a fusion to redox-sensitive yellow fluorescent protein.</title><author><name sortKey="Bjornberg, Olof" sort="Bjornberg, Olof" uniqKey="Bjornberg O" first="Olof" last="Björnberg">Olof Björnberg</name><affiliation wicri:level="1"><nlm:affiliation>Carlsberg Laboratory, DK-2500 Copenhagen Valby, Denmark.</nlm:affiliation><country xml:lang="fr">Danemark</country><wicri:regionArea>Carlsberg Laboratory, DK-2500 Copenhagen Valby</wicri:regionArea><wicri:noRegion>DK-2500 Copenhagen Valby</wicri:noRegion></affiliation></author><author><name sortKey=" Stergaard, Henrik" sort=" Stergaard, Henrik" uniqKey=" Stergaard H" first="Henrik" last=" Stergaard">Henrik Stergaard</name></author><author><name sortKey="Winther, Jakob R" sort="Winther, Jakob R" uniqKey="Winther J" first="Jakob R" last="Winther">Jakob R. Winther</name></author></analytic><series><title level="j">Biochemistry</title><idno type="ISSN">0006-2960</idno><imprint><date when="2006" type="published">2006</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Bacterial Proteins (metabolism)</term><term>Cystamine (chemistry)</term><term>Disulfides (chemistry)</term><term>Ethanol (analogs & derivatives)</term><term>Ethanol (chemistry)</term><term>Glutaredoxins (MeSH)</term><term>Glutathione Disulfide (metabolism)</term><term>Hydrogen Peroxide (chemistry)</term><term>Luminescent Proteins (metabolism)</term><term>Oxidation-Reduction (MeSH)</term><term>Oxidoreductases (genetics)</term><term>Oxidoreductases (metabolism)</term><term>Recombinant Fusion Proteins (metabolism)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Cystamine (composition chimique)</term><term>Disulfure de glutathion (métabolisme)</term><term>Disulfures (composition chimique)</term><term>Glutarédoxines (MeSH)</term><term>Oxidoreductases (génétique)</term><term>Oxidoreductases (métabolisme)</term><term>Oxydoréduction (MeSH)</term><term>Peroxyde d'hydrogène (composition chimique)</term><term>Protéines bactériennes (métabolisme)</term><term>Protéines de fusion recombinantes (métabolisme)</term><term>Protéines luminescentes (métabolisme)</term><term>Éthanol (analogues et dérivés)</term><term>Éthanol (composition chimique)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="analogs & derivatives" xml:lang="en"><term>Ethanol</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Cystamine</term><term>Disulfides</term><term>Ethanol</term><term>Hydrogen Peroxide</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Oxidoreductases</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Bacterial Proteins</term><term>Glutathione Disulfide</term><term>Luminescent Proteins</term><term>Oxidoreductases</term><term>Recombinant Fusion Proteins</term></keywords><keywords scheme="MESH" type="chemical" xml:lang="en"><term>Glutaredoxins</term></keywords><keywords scheme="MESH" qualifier="analogues et dérivés" xml:lang="fr"><term>Éthanol</term></keywords><keywords scheme="MESH" qualifier="composition chimique" xml:lang="fr"><term>Cystamine</term><term>Disulfures</term><term>Peroxyde d'hydrogène</term><term>Éthanol</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Oxidoreductases</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Disulfure de glutathion</term><term>Oxidoreductases</term><term>Protéines bactériennes</term><term>Protéines de fusion recombinantes</term><term>Protéines luminescentes</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Oxidation-Reduction</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Glutarédoxines</term><term>Oxydoréduction</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Redox-sensitive yellow fluorescent protein (rxYFP) contains a dithiol disulfide pair that is thermodynamically suitable for monitoring intracellular glutathione redox potential. Glutaredoxin 1 (Grx1p) from yeast is known to catalyze the redox equilibrium between rxYFP and glutathione, and here, we have generated a fusion of the two proteins, rxYFP-Grx1p. In comparison to isolated subunits, intramolecular transfer of reducing equivalents made the fusion protein kinetically superior in reactions with glutathione. The rate of GSSG oxidation was thus improved by a factor of 3300. The reaction with GSSG most likely takes place entirely through a glutathionylated intermediate and not through transfer of an intramolecular disulfide bond. However, during oxidation by H(2)O(2), hydroxyethyl disulfide, or cystine, the glutaredoxin domain reacted first, followed by a rate-limiting (0.13 min(-)(1)) transfer of a disulfide bond to the other domain. Thus, reactivity toward other oxidants remains low, giving almost absolute glutathione specificity. We have further studied CPYC --> CPYS variants in the active site of Grx1p and found that the single Cys variant had elevated oxidoreductase activity separately and in the fusion. This could not be ascribed to the lack of an unproductive side reaction to glutaredoxin disulfide. Instead, slower alkylation kinetics with iodoacetamide indicates a better leaving-group capability of the remaining cysteine residue, which can explain the increased activity.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">16475825</PMID><DateCompleted><Year>2006</Year><Month>04</Month><Day>10</Day></DateCompleted><DateRevised><Year>2016</Year><Month>11</Month><Day>24</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">0006-2960</ISSN><JournalIssue CitedMedium="Print"><Volume>45</Volume><Issue>7</Issue><PubDate><Year>2006</Year><Month>Feb</Month><Day>21</Day></PubDate></JournalIssue><Title>Biochemistry</Title><ISOAbbreviation>Biochemistry</ISOAbbreviation></Journal><ArticleTitle>Mechanistic insight provided by glutaredoxin within a fusion to redox-sensitive yellow fluorescent protein.</ArticleTitle><Pagination><MedlinePgn>2362-71</MedlinePgn></Pagination><Abstract><AbstractText>Redox-sensitive yellow fluorescent protein (rxYFP) contains a dithiol disulfide pair that is thermodynamically suitable for monitoring intracellular glutathione redox potential. Glutaredoxin 1 (Grx1p) from yeast is known to catalyze the redox equilibrium between rxYFP and glutathione, and here, we have generated a fusion of the two proteins, rxYFP-Grx1p. In comparison to isolated subunits, intramolecular transfer of reducing equivalents made the fusion protein kinetically superior in reactions with glutathione. The rate of GSSG oxidation was thus improved by a factor of 3300. The reaction with GSSG most likely takes place entirely through a glutathionylated intermediate and not through transfer of an intramolecular disulfide bond. However, during oxidation by H(2)O(2), hydroxyethyl disulfide, or cystine, the glutaredoxin domain reacted first, followed by a rate-limiting (0.13 min(-)(1)) transfer of a disulfide bond to the other domain. Thus, reactivity toward other oxidants remains low, giving almost absolute glutathione specificity. We have further studied CPYC --> CPYS variants in the active site of Grx1p and found that the single Cys variant had elevated oxidoreductase activity separately and in the fusion. This could not be ascribed to the lack of an unproductive side reaction to glutaredoxin disulfide. Instead, slower alkylation kinetics with iodoacetamide indicates a better leaving-group capability of the remaining cysteine residue, which can explain the increased activity.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Björnberg</LastName><ForeName>Olof</ForeName><Initials>O</Initials><AffiliationInfo><Affiliation>Carlsberg Laboratory, DK-2500 Copenhagen Valby, Denmark.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Østergaard</LastName><ForeName>Henrik</ForeName><Initials>H</Initials></Author><Author ValidYN="Y"><LastName>Winther</LastName><ForeName>Jakob R</ForeName><Initials>JR</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Biochemistry</MedlineTA><NlmUniqueID>0370623</NlmUniqueID><ISSNLinking>0006-2960</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D001426">Bacterial Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D004220">Disulfides</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D054477">Glutaredoxins</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D008164">Luminescent Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D011993">Recombinant Fusion Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="C054562">yellow fluorescent protein, Bacteria</NameOfSubstance></Chemical><Chemical><RegistryNumber>3K9958V90M</RegistryNumber><NameOfSubstance UI="D000431">Ethanol</NameOfSubstance></Chemical><Chemical><RegistryNumber>45543L74BS</RegistryNumber><NameOfSubstance UI="C031319">2-hydroxyethyl disulfide</NameOfSubstance></Chemical><Chemical><RegistryNumber>BBX060AN9V</RegistryNumber><NameOfSubstance UI="D006861">Hydrogen Peroxide</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 1.-</RegistryNumber><NameOfSubstance UI="D010088">Oxidoreductases</NameOfSubstance></Chemical><Chemical><RegistryNumber>R110LV8L02</RegistryNumber><NameOfSubstance UI="D003538">Cystamine</NameOfSubstance></Chemical><Chemical><RegistryNumber>ULW86O013H</RegistryNumber><NameOfSubstance UI="D019803">Glutathione Disulfide</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D001426" MajorTopicYN="N">Bacterial Proteins</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D003538" MajorTopicYN="N">Cystamine</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D004220" MajorTopicYN="N">Disulfides</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D000431" MajorTopicYN="N">Ethanol</DescriptorName><QualifierName UI="Q000031" MajorTopicYN="N">analogs & derivatives</QualifierName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D054477" MajorTopicYN="N">Glutaredoxins</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D019803" MajorTopicYN="N">Glutathione Disulfide</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D006861" MajorTopicYN="N">Hydrogen Peroxide</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D008164" MajorTopicYN="N">Luminescent Proteins</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D010084" MajorTopicYN="N">Oxidation-Reduction</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D010088" MajorTopicYN="N">Oxidoreductases</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D011993" MajorTopicYN="N">Recombinant Fusion Proteins</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="pubmed"><Year>2006</Year><Month>2</Month><Day>16</Day><Hour>9</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2006</Year><Month>4</Month><Day>11</Day><Hour>9</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2006</Year><Month>2</Month><Day>16</Day><Hour>9</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">16475825</ArticleId><ArticleId IdType="doi">10.1021/bi0522495</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>Danemark</li></country></list><tree><noCountry><name sortKey=" Stergaard, Henrik" sort=" Stergaard, Henrik" uniqKey=" Stergaard H" first="Henrik" last=" Stergaard">Henrik Stergaard</name><name sortKey="Winther, Jakob R" sort="Winther, Jakob R" uniqKey="Winther J" first="Jakob R" last="Winther">Jakob R. Winther</name></noCountry><country name="Danemark"><noRegion><name sortKey="Bjornberg, Olof" sort="Bjornberg, Olof" uniqKey="Bjornberg O" first="Olof" last="Björnberg">Olof Björnberg</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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