Protein self-assembly and lipid binding in the folding of the potassium channel KcsA.
Moderate concentrations of the alcohol 2,2,2-trifluoroethanol (TFE) cause the coupled unfolding and dissociation into subunits of the homotetrameric potassium channel KcsA, in a process that is partially irreversible when the protein is solubilized in plain dodecyl beta-d-maltoside (DDM) micelles [Barrera et al. (2005) Biochemistry 44, 14344-52]. Here we report that the transition from the folded tetramer to the unfolded monomer becomes completely reversible when KcsA is solubilized in mixed micelles composed of the detergent DDM and the lipids DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) and DOPG (1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]). This result suggests that lipids may act as effectors in the tetramerization of KcsA. The observed reversibility allowed the determination of the standard free energy of the folding reaction of KcsA: DeltaG = 30.5 +/- 3.1 kcal x mol-1. We also observed that, prior to the unfolding of the tetramer, the presence of lower TFE concentrations causes the disassembly of supramolecular clusters of KcsA into the individual tetrameric molecules. Within the limits of experimental resolution, this is also a reversible process, but unlike the tetramer to monomer transition from above, the level of clustering is not influenced by the presence of solubilized lipids. These observations suggest a distinct role of the lipids in the different in vitro assembly steps (folding/tetramerization and clustering) of KcsA.
Barrera
Francisco N
FN
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche, 03202 Alicante, Spain.
Renart
M Lourdes
ML
Poveda
José A
JA
de Kruijff
Ben
B
Killian
J Antoinette
JA
González-Ros
José M
JM
D001426:Bacterial Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D050356:Lipid Metabolism; D015221:Potassium Channels / Q000737:chemistry / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014270:Trifluoroethanol / Q000737:chemistry
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C504448:KcsA protein, Streptomyces coelicolor; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D014270:Trifluoroethanol