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dc.contributor.authorGarcia, Alvaro
dc.contributor.authorPromod, Pratap R.
dc.contributor.authorLupfert, Christian
dc.contributor.authorCornelius, Flemming
dc.contributor.authorJacquemin, Denis
dc.contributor.authorLev, Bogdan
dc.contributor.authorAllen, Toby W.
dc.contributor.authorClarke, Ronald J.
dc.date.accessioned2019-09-10T02:15:47Z
dc.date.available2019-09-10T02:15:47Z
dc.date.issued2017-01-20
dc.identifier.citationGarcia, A., Pratap, P. R., Lüpfert, C., Cornelius, F., Jacquemin, D., Lev, B., … Clarke, R. J. (2017). The voltage-sensitive dye RH421 detects a Na + ,K + -ATPase conformational change at the membrane surface. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1859(5), 813–823. https://doi.org/10.1016/j.bbamem.2017.01.022en_AU
dc.identifier.urihttp://hdl.handle.net/2123/21063
dc.description.abstractRH421 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.en_AU
dc.description.sponsorshipAustralian Research Councilen_AU
dc.language.isoen_AUen_AU
dc.publisherElsevieren_AU
dc.relationARC DP121003548, ARC DP150101112, ARC DP170101732, NHMRC APP1104259en_AU
dc.rights© 2017. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/en_AU
dc.subjectRH421 fluorescenceen_AU
dc.subjectNa+,K+-ATPaseen_AU
dc.subjectstopped-flow kineticsen_AU
dc.subjectmolecular dynamicsen_AU
dc.subjectN-terminusen_AU
dc.subjectphosphorylationen_AU
dc.titleThe voltage-sensitive dye RH421 detects a Na+,K+-ATPase conformational change at the membrane interfaceen_AU
dc.typeArticleen_AU
dc.subject.asrcFoR::030403 - Characterisation of Biological Macromoleculesen_AU
dc.identifier.doidx.doi.org/10.1016/j.bbamem.2017.01.022
dc.type.pubtypePost-printen_AU


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