Gramicidins A, B, and C are the three most abundant, naturally occurring analogues of this family of channel-forming antibiotic. GB and GC differ from the parent pentadecapeptide, GA, by single residue mutations, W11F and W11Y, respectively. Although these mutations occur in the cation binding region of the channel, they do not affect monovalent cation specificity, but are known to alter cation-binding affinities, thermodynamic parameters of cation binding, conductance and the activation energy for ion transport. The structures of all three analogues incorporated into deuterated sodium dodecyl sulfate micelles have been obtained using solution state 2D-NMR spectroscopy and molecular modeling. For the first time, a rigorous comparison of the 3D structures of these analogues reveals that the amino acid substitutions do not have a significant effect on backbone conformation, thus eliminating channel differences as the cause of variations in transport properties. Variable positions of methyl groups in valine and leucine residues have been linked to molecular motions and are not likely to affect ion flow through the channel. Thus, it is concluded that changes in the magnitude and orientation of the dipole moment at residue 11 are responsible for altering monovalent cation transport.

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To further investigate the effect of single amino acid substitution on the structure and function of the gramicidin channel, an analogue of gramicidin A (GA) has been synthesized in which Trp(15) is replaced by Gly in the critical aqueous interface and cation binding region. The structure of Gly(15)-GA incorporated into SDS micelles has been determined using a combination of 2D-NMR spectroscopy and molecular modeling. Like the parent GA, Gly(15)-GA forms a dimeric channel composed of two single-stranded, right-handed beta(6.3)-helices joined by hydrogen bonds between their N-termini. The replacement of Trp(15) by Gly does not have a significant effect on backbone structure or side chain conformations with the exception of Trp(11) in which the indole ring is rotated away from the channel axis. Measurement of the equilibrium binding constants and Delta G for the binding of monovalent cations to GA and Gly(15)-GA channels incorporated into PC vesicles using (205)Tl NMR spectroscopy shows that monovalent cations bind much more weakly to the Gly(15)-GA channel entrance than to GA channels. Utilizing the magnetization inversion transfer NMR technique, the transport of Na(+) ions through GA and Gly(15)-GA channels incorporated into PC/PG vesicles has been investigated. The Gly(15) substitution produces an increase in the activation enthalpy of transport and thus a significant decrease in the transport rate of the Na(+) ion is observed. The single-channel appearances show that the conducting channels have a single, well-defined structure. Consistent with the NMR results, the single-channel conductances are reduced by 30% and the lifetimes by 70%. It is concluded that the decrease in cation binding, transport, and conductance in Gly(15)-GA results from the removal of the Trp(15) dipole and, to a lesser extent, the change in orientation of Trp(11).

We report a 67Zn nuclear magnetic resonance (NMR) study of compounds containing zinc ions coordinated by oxygen, nitrogen, and sulfur ligands. New information concerning 67Zn nuclear quadrupole coupling constants (NQCC) and chemical shift was obtained from magic-angle spinning (MAS) spectra of solid compounds containing both natural abundance and enriched 67Zn isotopes. Rapid ligand exchange processes of [Zn(thiourea)4]2+ in aqueous solutions were also observed.Key words: 67Zn NMR, nuclear quadrupolar coupling constant, ligand exchange.