The voltage-sensitive dye RH421 detects a Na(+),K(+)-ATPase conformational change at the membrane surface.
Identifieur interne : 000C54 ( PubMed/Corpus ); précédent : 000C53; suivant : 000C55The voltage-sensitive dye RH421 detects a Na(+),K(+)-ATPase conformational change at the membrane surface.
Auteurs : Alvaro Garcia ; Promod R. Pratap ; Christian Lüpfert ; Flemming Cornelius ; Denis Jacquemin ; Bogdan Lev ; Toby W. Allen ; Ronald J. ClarkeSource :
- Biochimica et biophysica acta [ 0006-3002 ] ; 2017.
English descriptors
- KwdEn :
- MESH :
- chemical , chemistry : Lipid Bilayers, Polylysine, Sodium-Potassium-Exchanging ATPase.
- chemical : Fluorescent Dyes.
- Molecular Dynamics Simulation, Phosphorylation, Protein Conformation, Sequence Analysis, Protein.
Abstract
RH421 is a voltage-sensitive fluorescent styrylpyridinium dye which has often been used to probe the kinetics of Na(+),K(+)-ATPase partial reactions. The origin of the dye's response has up to now been unclear. Here we show that RH421 responds to phosphorylation of the Na(+),K(+)-ATPase by inorganic phosphate with a fluorescence increase. Analysis of the kinetics of the fluorescence response indicates that the probe is not detecting phosphorylation itself but rather a shift in the protein's E1/E2 conformational equilibrium induced by preferential phosphate binding to and phosphorylation of enzyme in the E2 conformation. Molecular dynamics simulations of crystal structures in lipid bilayers indicate some change in the protein's hydrophobic thickness during the E1-E2 transition, which may influence the dye response. However, the transition is known to involve significant rearrangement of the protein's highly charged lysine-rich cytoplasmic N-terminal sequence. Using poly-l-lysine as a model of the N-terminus, we show that an analogous response of RH421 to the E1→E2P conformational change is produced by poly-l-lysine binding to the surface of the Na(+),K(+)-ATPase-containing membrane fragments. Thus, it seems that the prime origin of the RH421 fluorescence response is a change in the interaction of the protein's N-terminus with the surrounding membrane. Quantum mechanical calculations of the dye's visible absorption spectrum give further support to this conclusion. The results obtained indicate that membrane binding and release of the N-terminus of the Na(+),K(+)-ATPase α-subunit are intimately involved in the protein's catalytic cycle and could represent an effective site of regulation.
DOI: 10.1016/j.bbamem.2017.01.022
PubMed: 28115116
Links to Exploration step
pubmed:28115116Le document en format XML
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Pratap</name><affiliation><nlm:affiliation>Department of Physics and Astronomy, University of North Carolina at Greensboro, Greensboro, NC 27402-6170, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Lupfert, Christian" sort="Lupfert, Christian" uniqKey="Lupfert C" first="Christian" last="Lüpfert">Christian Lüpfert</name><affiliation><nlm:affiliation>Department of Biophysical Chemistry, Max-Planck-Institute of Biophysics, D-60438 Frankfurt am Main, Germany.</nlm:affiliation></affiliation></author><author><name sortKey="Cornelius, Flemming" sort="Cornelius, Flemming" uniqKey="Cornelius F" first="Flemming" last="Cornelius">Flemming Cornelius</name><affiliation><nlm:affiliation>Department of Biomedicine, University of Aarhus, DK-8000, Aarhus C, Denmark.</nlm:affiliation></affiliation></author><author><name sortKey="Jacquemin, Denis" sort="Jacquemin, Denis" uniqKey="Jacquemin D" first="Denis" last="Jacquemin">Denis Jacquemin</name><affiliation><nlm:affiliation>CEISAM Lab, UMR CNRS 6230, Science and Technology Faculty, BP92208, University of Nantes, 44322 Nantes Cedex 3, France; Institut Universitaire de France, 1, Rue Descartes, 75231, Paris, France.</nlm:affiliation></affiliation></author><author><name sortKey="Lev, Bogdan" sort="Lev, Bogdan" uniqKey="Lev B" first="Bogdan" last="Lev">Bogdan Lev</name><affiliation><nlm:affiliation>School of Applied Science and Health Innovations Research Institute, RMIT University, Melbourne, Australia.</nlm:affiliation></affiliation></author><author><name sortKey="Allen, Toby W" sort="Allen, Toby W" uniqKey="Allen T" first="Toby W" last="Allen">Toby W. Allen</name><affiliation><nlm:affiliation>School of Applied Science and Health Innovations Research Institute, RMIT University, Melbourne, Australia; Department of Chemistry, University of California, Davis, CA 95616, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Clarke, Ronald J" sort="Clarke, Ronald J" uniqKey="Clarke R" first="Ronald J" last="Clarke">Ronald J. Clarke</name><affiliation><nlm:affiliation>School of Chemistry, University of Sydney, Sydney, NSW 2006, Australia. Electronic address: ronald.clarke@sydney.edu.au.</nlm:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2017">2017</date><idno type="RBID">pubmed:28115116</idno><idno type="pmid">28115116</idno><idno type="doi">10.1016/j.bbamem.2017.01.022</idno><idno type="wicri:Area/PubMed/Corpus">000C54</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">000C54</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">The voltage-sensitive dye RH421 detects a Na(+),K(+)-ATPase conformational change at the membrane surface.</title><author><name sortKey="Garcia, Alvaro" sort="Garcia, Alvaro" uniqKey="Garcia A" first="Alvaro" last="Garcia">Alvaro Garcia</name><affiliation><nlm:affiliation>School of Chemistry, University of Sydney, Sydney, NSW 2006, Australia.</nlm:affiliation></affiliation></author><author><name sortKey="Pratap, Promod R" sort="Pratap, Promod R" uniqKey="Pratap P" first="Promod R" last="Pratap">Promod R. Pratap</name><affiliation><nlm:affiliation>Department of Physics and Astronomy, University of North Carolina at Greensboro, Greensboro, NC 27402-6170, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Lupfert, Christian" sort="Lupfert, Christian" uniqKey="Lupfert C" first="Christian" last="Lüpfert">Christian Lüpfert</name><affiliation><nlm:affiliation>Department of Biophysical Chemistry, Max-Planck-Institute of Biophysics, D-60438 Frankfurt am Main, Germany.</nlm:affiliation></affiliation></author><author><name sortKey="Cornelius, Flemming" sort="Cornelius, Flemming" uniqKey="Cornelius F" first="Flemming" last="Cornelius">Flemming Cornelius</name><affiliation><nlm:affiliation>Department of Biomedicine, University of Aarhus, DK-8000, Aarhus C, Denmark.</nlm:affiliation></affiliation></author><author><name sortKey="Jacquemin, Denis" sort="Jacquemin, Denis" uniqKey="Jacquemin D" first="Denis" last="Jacquemin">Denis Jacquemin</name><affiliation><nlm:affiliation>CEISAM Lab, UMR CNRS 6230, Science and Technology Faculty, BP92208, University of Nantes, 44322 Nantes Cedex 3, France; Institut Universitaire de France, 1, Rue Descartes, 75231, Paris, France.</nlm:affiliation></affiliation></author><author><name sortKey="Lev, Bogdan" sort="Lev, Bogdan" uniqKey="Lev B" first="Bogdan" last="Lev">Bogdan Lev</name><affiliation><nlm:affiliation>School of Applied Science and Health Innovations Research Institute, RMIT University, Melbourne, Australia.</nlm:affiliation></affiliation></author><author><name sortKey="Allen, Toby W" sort="Allen, Toby W" uniqKey="Allen T" first="Toby W" last="Allen">Toby W. Allen</name><affiliation><nlm:affiliation>School of Applied Science and Health Innovations Research Institute, RMIT University, Melbourne, Australia; Department of Chemistry, University of California, Davis, CA 95616, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Clarke, Ronald J" sort="Clarke, Ronald J" uniqKey="Clarke R" first="Ronald J" last="Clarke">Ronald J. Clarke</name><affiliation><nlm:affiliation>School of Chemistry, University of Sydney, Sydney, NSW 2006, Australia. Electronic address: ronald.clarke@sydney.edu.au.</nlm:affiliation></affiliation></author></analytic><series><title level="j">Biochimica et biophysica acta</title><idno type="ISSN">0006-3002</idno><imprint><date when="2017" type="published">2017</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Fluorescent Dyes</term><term>Lipid Bilayers (chemistry)</term><term>Molecular Dynamics Simulation</term><term>Phosphorylation</term><term>Polylysine (chemistry)</term><term>Protein Conformation</term><term>Sequence Analysis, Protein</term><term>Sodium-Potassium-Exchanging ATPase (chemistry)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Lipid Bilayers</term><term>Polylysine</term><term>Sodium-Potassium-Exchanging ATPase</term></keywords><keywords scheme="MESH" type="chemical" xml:lang="en"><term>Fluorescent Dyes</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Molecular Dynamics Simulation</term><term>Phosphorylation</term><term>Protein Conformation</term><term>Sequence Analysis, Protein</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">RH421 is a voltage-sensitive fluorescent styrylpyridinium dye which has often been used to probe the kinetics of Na(+),K(+)-ATPase partial reactions. The origin of the dye's response has up to now been unclear. Here we show that RH421 responds to phosphorylation of the Na(+),K(+)-ATPase by inorganic phosphate with a fluorescence increase. Analysis of the kinetics of the fluorescence response indicates that the probe is not detecting phosphorylation itself but rather a shift in the protein's E1/E2 conformational equilibrium induced by preferential phosphate binding to and phosphorylation of enzyme in the E2 conformation. Molecular dynamics simulations of crystal structures in lipid bilayers indicate some change in the protein's hydrophobic thickness during the E1-E2 transition, which may influence the dye response. However, the transition is known to involve significant rearrangement of the protein's highly charged lysine-rich cytoplasmic N-terminal sequence. Using poly-l-lysine as a model of the N-terminus, we show that an analogous response of RH421 to the E1→E2P conformational change is produced by poly-l-lysine binding to the surface of the Na(+),K(+)-ATPase-containing membrane fragments. Thus, it seems that the prime origin of the RH421 fluorescence response is a change in the interaction of the protein's N-terminus with the surrounding membrane. Quantum mechanical calculations of the dye's visible absorption spectrum give further support to this conclusion. The results obtained indicate that membrane binding and release of the N-terminus of the Na(+),K(+)-ATPase α-subunit are intimately involved in the protein's catalytic cycle and could represent an effective site of regulation.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">28115116</PMID><DateCreated><Year>2017</Year><Month>01</Month><Day>24</Day></DateCreated><DateCompleted><Year>2017</Year><Month>08</Month><Day>31</Day></DateCompleted><DateRevised><Year>2017</Year><Month>08</Month><Day>31</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Print">0006-3002</ISSN><JournalIssue CitedMedium="Print"><Volume>1859</Volume><Issue>5</Issue><PubDate><Year>2017</Year><Month>05</Month></PubDate></JournalIssue><Title>Biochimica et biophysica acta</Title><ISOAbbreviation>Biochim. Biophys. Acta</ISOAbbreviation></Journal><ArticleTitle>The voltage-sensitive dye RH421 detects a Na(+),K(+)-ATPase conformational change at the membrane surface.</ArticleTitle><Pagination><MedlinePgn>813-823</MedlinePgn></Pagination><ELocationID EIdType="pii" ValidYN="Y">S0005-2736(17)30030-5</ELocationID><ELocationID EIdType="doi" ValidYN="Y">10.1016/j.bbamem.2017.01.022</ELocationID><Abstract><AbstractText>RH421 is a voltage-sensitive fluorescent styrylpyridinium dye which has often been used to probe the kinetics of Na(+),K(+)-ATPase partial reactions. The origin of the dye's response has up to now been unclear. Here we show that RH421 responds to phosphorylation of the Na(+),K(+)-ATPase by inorganic phosphate with a fluorescence increase. Analysis of the kinetics of the fluorescence response indicates that the probe is not detecting phosphorylation itself but rather a shift in the protein's E1/E2 conformational equilibrium induced by preferential phosphate binding to and phosphorylation of enzyme in the E2 conformation. Molecular dynamics simulations of crystal structures in lipid bilayers indicate some change in the protein's hydrophobic thickness during the E1-E2 transition, which may influence the dye response. However, the transition is known to involve significant rearrangement of the protein's highly charged lysine-rich cytoplasmic N-terminal sequence. Using poly-l-lysine as a model of the N-terminus, we show that an analogous response of RH421 to the E1→E2P conformational change is produced by poly-l-lysine binding to the surface of the Na(+),K(+)-ATPase-containing membrane fragments. Thus, it seems that the prime origin of the RH421 fluorescence response is a change in the interaction of the protein's N-terminus with the surrounding membrane. Quantum mechanical calculations of the dye's visible absorption spectrum give further support to this conclusion. The results obtained indicate that membrane binding and release of the N-terminus of the Na(+),K(+)-ATPase α-subunit are intimately involved in the protein's catalytic cycle and could represent an effective site of regulation.</AbstractText><CopyrightInformation>Copyright © 2017 Elsevier B.V. All rights reserved.</CopyrightInformation></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Garcia</LastName><ForeName>Alvaro</ForeName><Initials>A</Initials><AffiliationInfo><Affiliation>School of Chemistry, University of Sydney, Sydney, NSW 2006, Australia.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Pratap</LastName><ForeName>Promod R</ForeName><Initials>PR</Initials><AffiliationInfo><Affiliation>Department of Physics and Astronomy, University of North Carolina at Greensboro, Greensboro, NC 27402-6170, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Lüpfert</LastName><ForeName>Christian</ForeName><Initials>C</Initials><AffiliationInfo><Affiliation>Department of Biophysical Chemistry, Max-Planck-Institute of Biophysics, D-60438 Frankfurt am Main, Germany.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Cornelius</LastName><ForeName>Flemming</ForeName><Initials>F</Initials><AffiliationInfo><Affiliation>Department of Biomedicine, University of Aarhus, DK-8000, Aarhus C, Denmark.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Jacquemin</LastName><ForeName>Denis</ForeName><Initials>D</Initials><AffiliationInfo><Affiliation>CEISAM Lab, UMR CNRS 6230, Science and Technology Faculty, BP92208, University of Nantes, 44322 Nantes Cedex 3, France; Institut Universitaire de France, 1, Rue Descartes, 75231, Paris, France.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Lev</LastName><ForeName>Bogdan</ForeName><Initials>B</Initials><AffiliationInfo><Affiliation>School of Applied Science and Health Innovations Research Institute, RMIT University, Melbourne, Australia.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Allen</LastName><ForeName>Toby W</ForeName><Initials>TW</Initials><AffiliationInfo><Affiliation>School of Applied Science and Health Innovations Research Institute, RMIT University, Melbourne, Australia; Department of Chemistry, University of California, Davis, CA 95616, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Clarke</LastName><ForeName>Ronald J</ForeName><Initials>RJ</Initials><AffiliationInfo><Affiliation>School of Chemistry, University of Sydney, Sydney, NSW 2006, Australia. Electronic address: ronald.clarke@sydney.edu.au.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2017</Year><Month>01</Month><Day>20</Day></ArticleDate></Article><MedlineJournalInfo><Country>Netherlands</Country><MedlineTA>Biochim Biophys Acta</MedlineTA><NlmUniqueID>0217513</NlmUniqueID><ISSNLinking>0006-3002</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D005456">Fluorescent Dyes</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D008051">Lipid Bilayers</NameOfSubstance></Chemical><Chemical><RegistryNumber>25104-18-1</RegistryNumber><NameOfSubstance UI="D011107">Polylysine</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 3.6.3.9</RegistryNumber><NameOfSubstance UI="D000254">Sodium-Potassium-Exchanging ATPase</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D005456" MajorTopicYN="Y">Fluorescent Dyes</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D008051" MajorTopicYN="N">Lipid Bilayers</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="Y">chemistry</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D056004" MajorTopicYN="N">Molecular Dynamics Simulation</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D010766" MajorTopicYN="N">Phosphorylation</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D011107" MajorTopicYN="N">Polylysine</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D011487" MajorTopicYN="N">Protein Conformation</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D020539" MajorTopicYN="N">Sequence Analysis, Protein</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D000254" MajorTopicYN="N">Sodium-Potassium-Exchanging ATPase</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="Y">chemistry</QualifierName></MeshHeading></MeshHeadingList><KeywordList Owner="NOTNLM"><Keyword MajorTopicYN="Y">Molecular dynamics</Keyword><Keyword MajorTopicYN="Y">N-terminus</Keyword><Keyword MajorTopicYN="Y">Na(+),K(+)-ATPase</Keyword><Keyword MajorTopicYN="Y">Phosphorylation</Keyword><Keyword MajorTopicYN="Y">RH421 fluorescence</Keyword><Keyword MajorTopicYN="Y">Stopped-flow kinetics</Keyword></KeywordList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="received"><Year>2016</Year><Month>10</Month><Day>19</Day></PubMedPubDate><PubMedPubDate PubStatus="revised"><Year>2017</Year><Month>01</Month><Day>09</Day></PubMedPubDate><PubMedPubDate PubStatus="accepted"><Year>2017</Year><Month>01</Month><Day>19</Day></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2017</Year><Month>1</Month><Day>25</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2017</Year><Month>9</Month><Day>1</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2017</Year><Month>1</Month><Day>25</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">28115116</ArticleId><ArticleId IdType="pii">S0005-2736(17)30030-5</ArticleId><ArticleId IdType="doi">10.1016/j.bbamem.2017.01.022</ArticleId></ArticleIdList></PubmedData></pubmed></record>Pour manipuler ce document sous Unix (Dilib)
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