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. 1989 Aug;56(2):307–314. doi: 10.1016/S0006-3495(89)82677-3

Determination of the structure of a membrane-incorporated ion channel. Solid-state nuclear magnetic resonance studies of gramicidin A.

R Smith 1, D E Thomas 1, F Separovic 1, A R Atkins 1, B A Cornell 1
PMCID: PMC1280480  PMID: 2476189

Abstract

Solid-state nuclear magnetic resonance (NMR) measurements on 13C-labeled analogues of the ion channel-forming peptide, gramicidin A, have been used to directly determine the structure of this peptide in lipid membranes. Seven gramicidin analogues, each labeled in a single carbonyl group of gly2, L-ala3, D-leu4, L-val7, D-leu10, D-leu12, or D-leu14 were synthesized by the solid-phase method. These gramicidin analogues were incorporated into aligned multilayers of dimyristoylphosphatidylcholine, or diether lipid bearing 14- or 16-carbon chains, at a 1:15 peptide:lipid mole ratio. Proton-enhanced, 13C, solid-state spectra were obtained at several temperatures and over a range of sample orientations with respect to the spectrometer magnetic field to permit accurate measurement of the chemical shift anisotropies. The observed anisotropies indicate that all of the labeled carbonyl bonds are oriented almost parallel to the molecular long axis and perpendicular to the lipid bilayer plane. These orientations are consistent with gramicidin forming a beta 6.3 single-strand helix that is oriented parallel to the methylene chains of the lipid molecules. Comparison of the linewidths from labeled residues that are in the innermost turn of the helix (gly2, ala3, and D-leu4), in the center of the molecule (val7), and in the turn nearest the lipid bilayer surface (D-leu10, D-leu12, and D-leu14) suggests that although the peptide behaves largely as a rigid barrel, segments of the peptide close to the membrane surface possess greater motional freedom. At temperatures above the gel-to-liquid crystalline transition temperature (Tc) the gramicidin molecules rotate, with a less than millisecond correlation time, about the bilayer normal: several degrees below Tc they become immobile on the NMR timescale, without change in the channel conformation. In the L beta' phase the linewidths of the D-leu10, D-leu'2, and D-leu" resonances become equal to those of the other labeled sites, indicating reduced but equivalent motion for all of the peptide carbonyl groups.

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Selected References

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