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Unfolding and refolding in vitro of a tetrameric, a-helical membrane protein: the prokaryotic potassium channel KcsA

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dc.contributor.authorBarrera Olivares, Francisco Nicolás
dc.contributor.authorRenart Pérez, María Lourdes
dc.contributor.authorMolina Gallego, María Luisa
dc.contributor.authorPoveda Larrosa, José Antonio
dc.contributor.authorEncinar Hidalgo, José Antonio
dc.contributor.authorFernández Carvajal, Asia María
dc.contributor.authorNeira Faleiro, José Luis
dc.contributor.authorGonzález Ros, José Manuel
dc.contributor.departmentBioquímica y Biología Molecular B e Inmunología
dc.date.accessioned2024-07-04T11:12:33Z
dc.date.available2024-07-04T11:12:33Z
dc.date.issued2005-10-06
dc.description© 2005 American Chemical Society. This document is the Published version of a Published Work that appeared in final form in Biochemistry. To access the final edited and published work see https://doi.org/10.1021/bi050845t
dc.description.abstract2,2,2-Trifluoroethanol (TFE) effectively destabilizes the otherwise highly stable tetrameric structure of the potassium channel KcsA, a predominantly α-helical membrane protein [Valiyaveetil, F. I., Zhou, Y., and MacKinnon, R. (2002) Biochemistry 41, 10771−10777]. Here, we report that the effects on the protein structure of increasing concentrations of TFE in detergent solution include two successive protein concentration-dependent, cooperative transitions. In the first of such transitions, occurring at lower TFE concentrations, the tetrameric KcsA simultaneously increases the exposure of tryptophan residues to the solvent, partly loses its secondary structure, and dissociates into its constituent subunits. Under these conditions, simple dilution of the TFE permits a highly efficient refolding and tetramerization of the protein in the detergent solution. Moreover, following reconstitution into asolectin giant liposomes, the refolded protein exhibits nativelike potassium channel activity, as assessed by patch-clamp methods. Conversely, the second cooperative transition occurring at higher TFE concentrations results in the irreversible denaturation of the protein. These results are interpreted in terms of a protein and TFE concentration-dependent reversible equilibrium between the folded tetrameric protein and partly unfolded monomeric subunits, in which folding and oligomerization (or unfolding and dissociation in the other direction of the equilibrium process) are seemingly coupled processes. At higher TFE concentrations this is followed by the irreversible conversion of the unfolded monomers into a denatured protein form.es
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dc.format.extent9es
dc.identifier.citationBiochemistry, 2005, Vol. 44, N. 43, pp. 14344–14352
dc.identifier.doihttps://doi.org/10.1021/bi050845t
dc.identifier.issnPrint: 0006-2960
dc.identifier.issnElectronic: 1520-4995
dc.identifier.urihttp://hdl.handle.net/10201/142855
dc.languageenges
dc.publisherAmerican Chemical Society
dc.relationThis work was supported by grants from the Spanish DGI BFI2002- 03410 (to J.M.G.-R.) and CTQ2004-04474 (to J.L.N.) and from the Agencia Valenciana de Ciencia y Tecnología 03/056 (to J.M.G.-R.) and 04/B402 (to J.L.N.). F.N.B. and M.L.R. were partly supported by predoctoral fellowships from the Ministerio de Educacio´n y Ciencia of Spain, while M.L.M. had a predoctoral fellowship from the Generalitat Valenciana.es
dc.relation.publisherversionhttps://pubs.acs.org/doi/10.1021/bi050845t#
dc.rights.accessRightsinfo:eu-repo/semantics/restrictedAccess
dc.subjectDissociationes
dc.subjectFluorescencees
dc.subjectMonomerses
dc.subjectPeptides and proteinses
dc.subjectPotassiumes
dc.titleUnfolding and refolding in vitro of a tetrameric, a-helical membrane protein: the prokaryotic potassium channel KcsAes
dc.typeinfo:eu-repo/semantics/articlees
dspace.entity.typePublicationes
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