SODIUM-ION BINDING IN THE GRAMICIDIN-A CHANNEL - SOLID-STATE NMR-STUDIES OF THE TRYPTOPHAN RESIDUES

Gramicidin A analogs, labeled with C-13 in the backbone carbonyl group s and the C-2 indole carbons of the tryptophan-11 and tryptophan-13 re sidues, were synthesized using t-Boc-protected amino acids. The purifi ed analogs were incorporated into phosphatidylcholine bilayers at a 1: 15 molar ratio and macroscopically aligned between glass coverslips. T he orientations of the labeled groups within the channel were investig ated using solid-state NMR and the effect of a monovalent ion (Na+) on the orientation of these groups determined. The presence of sodium io ns did not perturb the C-13 spectra of the tryptophan carbonyl groups. These results contrast with earlier results in which the Leu-10, Leu- 12, and Leu-14 carbonyl groups were found to be significantly affected by the presence of sodium ions and imply that the tryptophan carbonyl groups are not directly involved in ion binding. The channel form of gramicidin A has been demonstrated to be the right-handed form of the beta(6.3) helix: consequently, the tryptophan carbonyls would be direc ted away from the entrance to the channel and take part in internal hy drogen bonding, so that the presence of cations in the channel would h ave less effect than on the outer leucine residues. Sodium ions also h ad no effect on the C-2 indole resonance of the tryptophan side chains . However, a small change was observed in Trp-11 when the ether lipid, ditetradecylphosphatidylcholine, was substituted for the eater lipid, dimyristoylphosphatidylcholine, indicating some sensitivity of the gr amicidin side chains to the surrounding lipid.