Spectroscopic and functional characterization of the putative transmembrane segment of the minK potassium channel.
MinK (Isk) is a voltage-dependent K+ channel whose gene has been recently cloned and which consists of 130 amino acids [Takumi, T., Ohkubo, H., & Nakanishi, S. (1988) Science 242, 1042-1045]. The protein contains one putative transmembrane segment by hydropathy analysis. Whether this putative transmembrane segment is involved in the function of the protein was studied. A 32 amino acid peptide (residues 41-72) with the sequence SKLEALYILMVLGFFGFFTLGIMLSYIRSKKL, containing the hypothesized transmembrane domain, designed TM-minK, was synthesized and fluorescently labeled. The alpha-helical content of TM-minK, assessed in methanol using circular dichroism (CD), was 57%. The fluorescent emission spectrum of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled TM-minK displayed a blue shift upon binding to small unilamellar vesicles (SUV), reflecting a relocation of the fluorescent probe to an environment of increased apolarity, i.e., within the lipid bilayer. The increase in NBD's fluorescence upon mixing NBD-labeled TM-minK with small unilamellar vesicles (SUV) was used to generate a binding isotherm, from which was derived a surface partition coefficient of 5.5 x 10(4) M-1. Fluorescence energy transfer measurements between carboxyfluoresceine-labeled and rhodamine-labeled analogues suggest that TM-minK aggregates within membranes. In addition, single-channel experiments revealed that TM-minK can form single channels in planar lipid membranes only when a trans negative potential is applied. The findings herein experimentally support a role of the transmembrane segment of minK both in the assembly and as a constituent of the pore formed by the protein.
Ben-Efraim
I
I
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Bach
D
D
Shai
Y
Y
D009327:4-Chloro-7-nitrobenzofurazan / Q000737:chemistry; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000737:chemistry; D002942:Circular Dichroism; D008051:Lipid Bilayers; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D015221:Potassium Channels / Q000737:chemistry*; D024642:Potassium Channels, Voltage-Gated; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence
D008051:Lipid Bilayers; D008565:Membrane Proteins; D010446:Peptide Fragments; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; C059983:potassium channel protein I(sk); D009327:4-Chloro-7-nitrobenzofurazan