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Effects of Phenylalanine Substitutions in Gramicidin A on the Kinetics of Channel Formation in Vesicles and Channel Structure in SDS Micelles

Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas

Correspondence: Address reprint requests to J. F. Hinton, Dept. of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701. Tel.: 501-575-5143; Fax: 501-575-4049; E-mail: jhinton{at}uark.edu.

The common occurrence of Trp residues at the aqueous-lipid interface region of transmembrane channels is thought to be indicative of its importance for insertion and stabilization of the channel in membranes. To further investigate the effects of TrpPhe substitution on the structure and function of the gramicidin channel, four analogs of gramicidin A have been synthesized in which the tryptophan residues at positions 9, 11, 13, and 15 are sequentially replaced with phenylalanine. The three-dimensional structure of each viable analog has been determined using a combination of two-dimensional NMR techniques and distance geometry-simulated annealing structure calculations. These phenylalanine analogs adopt a homodimer motif, consisting of two ß^6.3 helices joined by six hydrogen bonds at their NH₂-termini. The replacement of the tryptophan residues does not have a significant effect on the backbone structure of the channels when compared to native gramicidin A, and only small effects are seen on side-chain conformations. Single-channel conductance measurements have shown that the conductance and lifetime of the channels are significantly affected by the replacement of the tryptophan residues (Wallace, 2000; Becker et al., 1991). The variation in conductance appears to be caused by the sequential removal of a tryptophan dipole, thereby removing the ion-dipole interaction at the channel entrance and at the ion binding site. Channel lifetime variations appear to be related to changing side chain-lipid interactions. This is supported by data relating to transport and incorporation kinetics.