The voltage-sensitive dye RH421 detects a Na(+),K(+)-ATPase conformational change at the membrane surface.
Identifieur interne : 000168 ( Main/Exploration ); précédent : 000167; suivant : 000169The voltage-sensitive dye RH421 detects a Na(+),K(+)-ATPase conformational change at the membrane surface.
Auteurs : Alvaro Garcia [Australie] ; Promod R. Pratap [États-Unis] ; Christian Lüpfert [Allemagne] ; Flemming Cornelius [Danemark] ; Denis Jacquemin [France] ; Bogdan Lev [Australie] ; Toby W. Allen [États-Unis] ; Ronald J. Clarke [Australie]Source :
- Biochimica et biophysica acta [ 0006-3002 ] ; 2017.
Descripteurs français
- KwdFr :
- MESH :
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
Affiliations:
- Allemagne, Australie, Danemark, France, États-Unis
- Californie, Caroline du Nord, District de Darmstadt, Hesse (Land), Nouvelle-Galles du Sud, Victoria (État), Île-de-France
- Francfort-sur-le-Main, Melbourne, Paris, Sydney
- Université de Sydney
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Le document en format XML
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Pratap</name><affiliation wicri:level="2"><nlm:affiliation>Department of Physics and Astronomy, University of North Carolina at Greensboro, Greensboro, NC 27402-6170, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Physics and Astronomy, University of North Carolina at Greensboro, Greensboro, NC 27402-6170</wicri:regionArea><placeName><region type="state">Caroline du Nord</region></placeName></affiliation></author><author><name sortKey="Lupfert, Christian" sort="Lupfert, Christian" uniqKey="Lupfert C" first="Christian" last="Lüpfert">Christian Lüpfert</name><affiliation wicri:level="3"><nlm:affiliation>Department of Biophysical Chemistry, Max-Planck-Institute of Biophysics, D-60438 Frankfurt am Main, Germany.</nlm:affiliation><country xml:lang="fr">Allemagne</country><wicri:regionArea>Department of Biophysical Chemistry, Max-Planck-Institute of Biophysics, D-60438 Frankfurt am Main</wicri:regionArea><placeName><region type="land" nuts="1">Hesse (Land)</region><region type="district" nuts="2">District de Darmstadt</region><settlement type="city">Francfort-sur-le-Main</settlement></placeName></affiliation></author><author><name sortKey="Cornelius, Flemming" sort="Cornelius, Flemming" uniqKey="Cornelius F" first="Flemming" last="Cornelius">Flemming Cornelius</name><affiliation wicri:level="1"><nlm:affiliation>Department of Biomedicine, University of Aarhus, DK-8000, Aarhus C, Denmark.</nlm:affiliation><country xml:lang="fr">Danemark</country><wicri:regionArea>Department of Biomedicine, University of Aarhus, DK-8000, Aarhus C</wicri:regionArea><wicri:noRegion>Aarhus C</wicri:noRegion></affiliation></author><author><name sortKey="Jacquemin, Denis" sort="Jacquemin, Denis" uniqKey="Jacquemin D" first="Denis" last="Jacquemin">Denis Jacquemin</name><affiliation wicri:level="3"><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><country xml:lang="fr">France</country><wicri:regionArea>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</wicri:regionArea><placeName><settlement type="city">Paris</settlement><region type="région" nuts="2">Île-de-France</region></placeName></affiliation></author><author><name sortKey="Lev, Bogdan" sort="Lev, Bogdan" uniqKey="Lev B" first="Bogdan" last="Lev">Bogdan Lev</name><affiliation wicri:level="3"><nlm:affiliation>School of Applied Science and Health Innovations Research Institute, RMIT University, Melbourne, Australia.</nlm:affiliation><country xml:lang="fr">Australie</country><wicri:regionArea>School of Applied Science and Health Innovations Research Institute, RMIT University, Melbourne</wicri:regionArea><placeName><settlement type="city">Melbourne</settlement><region type="état">Victoria (État)</region></placeName></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 wicri:level="2"><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><country xml:lang="fr">États-Unis</country><wicri:regionArea>School of Applied Science and Health Innovations Research Institute, RMIT University, Melbourne, Australia; Department of Chemistry, University of California, Davis, CA 95616</wicri:regionArea><placeName><region type="state">Californie</region></placeName></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 wicri:level="4"><nlm:affiliation>School of Chemistry, University of Sydney, Sydney, NSW 2006, Australia. Electronic address: ronald.clarke@sydney.edu.au.</nlm:affiliation><country xml:lang="fr">Australie</country><wicri:regionArea>School of Chemistry, University of Sydney, Sydney, NSW 2006</wicri:regionArea><orgName type="university">Université de Sydney</orgName><placeName><settlement type="city">Sydney</settlement><region type="état">Nouvelle-Galles du Sud</region></placeName></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="KwdFr" xml:lang="fr"><term>Analyse de séquence de protéine</term><term>Colorants fluorescents</term><term>Conformation des protéines</term><term>Double couche lipidique ()</term><term>Phosphorylation</term><term>Polylysine ()</term><term>Simulation de dynamique moléculaire</term><term>Sodium-Potassium-Exchanging ATPase ()</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><keywords scheme="MESH" xml:lang="fr"><term>Analyse de séquence de protéine</term><term>Colorants fluorescents</term><term>Conformation des protéines</term><term>Double couche lipidique</term><term>Phosphorylation</term><term>Polylysine</term><term>Simulation de dynamique moléculaire</term><term>Sodium-Potassium-Exchanging ATPase</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><affiliations><list><country><li>Allemagne</li><li>Australie</li><li>Danemark</li><li>France</li><li>États-Unis</li></country><region><li>Californie</li><li>Caroline du Nord</li><li>District de Darmstadt</li><li>Hesse (Land)</li><li>Nouvelle-Galles du Sud</li><li>Victoria (État)</li><li>Île-de-France</li></region><settlement><li>Francfort-sur-le-Main</li><li>Melbourne</li><li>Paris</li><li>Sydney</li></settlement><orgName><li>Université de Sydney</li></orgName></list><tree><country name="Australie"><region name="Nouvelle-Galles du Sud"><name sortKey="Garcia, Alvaro" sort="Garcia, Alvaro" uniqKey="Garcia A" first="Alvaro" last="Garcia">Alvaro Garcia</name></region><name sortKey="Clarke, Ronald J" sort="Clarke, Ronald J" uniqKey="Clarke R" first="Ronald J" last="Clarke">Ronald J. Clarke</name><name sortKey="Lev, Bogdan" sort="Lev, Bogdan" uniqKey="Lev B" first="Bogdan" last="Lev">Bogdan Lev</name></country><country name="États-Unis"><region name="Caroline du Nord"><name sortKey="Pratap, Promod R" sort="Pratap, Promod R" uniqKey="Pratap P" first="Promod R" last="Pratap">Promod R. Pratap</name></region><name sortKey="Allen, Toby W" sort="Allen, Toby W" uniqKey="Allen T" first="Toby W" last="Allen">Toby W. Allen</name></country><country name="Allemagne"><region name="Hesse (Land)"><name sortKey="Lupfert, Christian" sort="Lupfert, Christian" uniqKey="Lupfert C" first="Christian" last="Lüpfert">Christian Lüpfert</name></region></country><country name="Danemark"><noRegion><name sortKey="Cornelius, Flemming" sort="Cornelius, Flemming" uniqKey="Cornelius F" first="Flemming" last="Cornelius">Flemming Cornelius</name></noRegion></country><country name="France"><region name="Île-de-France"><name sortKey="Jacquemin, Denis" sort="Jacquemin, Denis" uniqKey="Jacquemin D" first="Denis" last="Jacquemin">Denis Jacquemin</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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