H-2 NUCLEAR-MAGNETIC-RESONANCE OF THE GRAMICIDIN-A BACKBONE IN A PHOSPHOLIPID-BILAYER

Solid-state H-2 NMR spectroscopy has been employed to study the channel conformation of gramicidin A (GA) in unoriented 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC) multilayers. Quadrupolar echo spectra were obtained at 44-degrees-C and 53-degrees-C, from gramicidin A labels in which the proton attached to the alpha-carbon of residue 3, 4, 5, 10, 12, or 14 was replaced with deuterium. Because of the nearly axially symmetric electric field gradient tensor, the quadrupolar splittings obtained from an unoriented multilamellar dispersion of DMPC and singly labeled GA directly yield unambiguous orientational constraints on the C-H-2 bonds.The average of the ratios of the quadrupolar splittings of the left-handed amino acids to those of the right-handed amino acids, <DELTA-nu-QL/DELTA-nu-QD>, is expected to be 0.97 +/- 0.04 for a relaxed right-handed beta-LD6,3 helix, while a ratio of 0.904 +/- 0.003 is expected for a left-handed beta-LD6,3 helix. Since we have experimentally determined this ratio to be 1.01 +/- 0.04, we conclude that the helix sense of the channel conformation of GA is right-handed. Assuming that the dominant motions are fast axial diffusion of the gramicidin molecule and reorientation of the diffusion axis with respect to the local bilayer normal, then the theoretical splittings may all be scaled down by a constant motional narrowing factor. In this case, a relaxed right-handed beta-LD6,3 helix, whose axis of motional averaging is roughly along the presumed helix axis, gave the best fit to experimental results. The reasonably uniform correspondence between the splittings predicted by the relaxed right-handed beta-LD6,3 helix and the observed splittings, for labels from both the inner and outer turn of GA, did not reflect a peptide backbone flexibility gradient, since an outer turn (i.e., the turn of the helix closest to the interface with the water) with greater flexibility would show additional motional narrowing for labels located there.