Conformation of the gramicidin A transmembrane channel: A 13C nuclear magnetic resonance study of 13C-enriched gramicidin in phosphatidylcholine vesicles

Abstract

We have determined the orientation and organization of the channel-forming polypeptide antibiotic, gramicidin A, in phosphatidylcholine vesicles using 13C nuclear magnetic resonance spectroscopy. This is the first direct structural evidence demonstrating the conformation of the molecule as found in membranes. The models previously proposed for the conformation of the dimer channel differ in the surface localization of the N and C termini. The N-terminal to N-terminal helical dimer has only the C termini on the surfaces of the membrane, while the C-terminal to C-terminal helical dimer has only the N termini on the surfaces. Parallel and antiparallel β-double helices have both the N and C termini on the surfaces. We have incorporated specific 13C nuclei at both the N and C termini of gramicidin and have used 13C chemical shifts and spin lattice relaxation time, T 1, measurements to determine the accessibility of these labels to three paramagnetic nuclear magnetic resonance probes. 1. (1) The water-soluble thulium ion causes a large change in chemical shift for the choline methyl groups, but little or no change for the fatty acid carbons even near the carbonyl end. The C-terminal gramicidin labels have changes in chemical shifts three- to fourfold greater than those of the N-terminal labels. 2. (2) The water-soluble manganous ion induces a T 1 rate enhancement for the fatty acid carbonyl that is no less than that for the choline methyls; while carbons 2 and 3 also have large rate enhancements, low values are obtained at the distal end of the chain. The C-terminal gramicidin label has a rate enhancement three- to fourfold greater than that of the N-terminal labels. 3. (3) Phosphatidylcholine labeled with a nitroxide spin label near the distal end of one chain induces T 1 rate enhancements in the distal 12 carbons of the fatty acid chain with little or no rate enhancement for the carbonyl and the choline head group. The gramicidin N-terminal valine methyls have the highest rate enhancement of any carbons in the experiment, while the C-terminal acetyl methyl has zero rate enhancement. All of our results indicate that the C-terminus of gramicidin in the channel is located near the surface of the membrane and the N-terminus is buried deep within the lipid bilayer. These findings strongly favor an N-terminal to N-terminal helical dimer, as originally proposed by Urry (1971), as the major conformation for the gramicidin channel in phosphatidylcholine vesicles.