Unfolding and refolding in vitro of a tetrameric, alpha-helical membrane protein: the prokaryotic potassium channel KcsA.
2,2,2-Trifluoroethanol (TFE) effectively destabilizes the otherwise highly stable tetrameric structure of the potassium channel KcsA, a predominantly alpha-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.
Barrera
Francisco N
FN
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche, 03202 Alicante, Spain.
Renart
M Lourdes
ML
Molina
M Luisa
ML
Poveda
José A
JA
Encinar
José A
JA
Fernández
Asia M
AM
Neira
José L
JL
González-Ros
José M
JM
D001426:Bacterial Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004594:Electrophysiology; D008565:Membrane Proteins / Q000737:chemistry*; D015221:Potassium Channels / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D014270:Trifluoroethanol / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry
D001426:Bacterial Proteins; D008565:Membrane Proteins; D015221:Potassium Channels; C107790:prokaryotic potassium channel; D012967:Sodium Dodecyl Sulfate; D014270:Trifluoroethanol; D014364:Tryptophan