Synthetic S-2 and H-5 segments of the Shaker K+ channel: secondary structure, membrane interaction, and assembly within phospholipid membranes.
Current models of voltage-activated K+ channels predict that the channels are formed by the coassembly of four polypeptide monomers, each of which consists of six transmembrane segments (S1-S6) and long terminal domains. The aqueous pores are thought to be composed of the conserved H-5 regions contributed by four monomers. In this study, two putative membrane-embedded segments of the Shaker K+ channel were synthesized. One segment corresponds to the putative, transmembrane helix S-2 (amino acids 275-300), and the other corresponds to the highly conserved 12 amino acid residues within the H-5 region [amino acids 432-443, designated (12)H-5]. Structural and functional characterization at elevated lipid/peptide molar ratios (> 3000:1) was performed on the two segments, as well as on a previously synthesized 21 amino acid long peptide with a sequence resembling the entire H-5 region (designated (21)H-5) (Peled & Shai, 1993). Circular dichroism spectroscopy revealed that S-2 adopts predominantly alpha-helical structure in both trifluoroethanol and 35 mM SDS (78% or 99%, respectively), while (12)H-5 and (21)H-5 adopt low alpha-helical structure only in the presence of 35 mM SDS. Functional characterization demonstrated that S-2 and (12)H-5 segments bind to zwitterionic phospholipids, with partition coefficients on the order of 10(4) M-1. Resonance energy transfer measurements, between donor/acceptor-labeled pairs of peptides, revealed that the peptides self-associate in their membrane-bound state, which may correlate with the existence of functional interactions between the conserved (12)H-5 regions of different subunits of K+ channels (Kirsch et al., 1993).(ABSTRACT TRUNCATED AT 250 WORDS)
Peled
H
H
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Shai
Y
Y
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D005456:Fluorescent Dyes; D008081:Liposomes; D008563:Membrane Lipids / Q000737:chemistry*; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000737:chemistry*; D015221:Potassium Channels / Q000138:chemical synthesis / Q000737:chemistry*; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D005456:Fluorescent Dyes; D008081:Liposomes; D008563:Membrane Lipids; D010743:Phospholipids; D015221:Potassium Channels; D014364:Tryptophan