Effects of glycine substitutions on the structure and function of gramicidin a channels

by Jordan, John Bradley; Shobana, S.; Andersen, Olaf S.; Hinton, James F.

Tryptophan residues often are found at the lipid-aqueous interface region of membrane-spanning proteins, including ion channels, where they are thought to be important determinants of protein structure and function. To better understand how Trp residues modulate the function of membrane-spanning channels, we have examined the effects of Trp replacements on the structure and function of gramicidin A channels. Analogues of gramicidin A in which the Trp residues at positions 9, 11, 13, and 15 were sequentially replaced with Gly were synthesized, and the three-dimensional structure of each analogue was determined using a combination of two-dimensional NMR techniques and distance geometry-simulated annealing structure calculations. Though Trp -> Gly substitutions destabilize the beta(6.3)-helical gA channel structure, it is possible to determine the structure of analogues with Trp -> Gly substitutions at positions 11, 13, and 15, but not for the analogue with the Trp -> Gly substitution at position 9. The Gly(11)-, Gly(13)-, and Gly(15)-gA analogues form channels that adopt a backbone fold identical to that of native gramicidin A, with only small changes in the side chain conformations of the unsubstituted residues. Single-channel current measurements show that the channel function and lifetime of the analogues are significantly affected by the Trp -> Gly replacements. The conductance variations appear to be caused by sequential removal of the Trp dipoles, which alter the ion-dipole interactions that modulate ion movement. The lifetime variations did not appear to follow a clear pattern.

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1520-4995; 0006-2960