CONFORMATION STATES OF GRAMICIDIN A ALONG THE PATHWAY TO THE FORMATION OF CHANNELS IN MODEL MEMBRANES DETERMINED BY 2D NMR AND CIRCULAR-DICHROISM SPECTROSCOPY

Gramicidin A incorporated into SDS (sodium dodecyl sulfate) micelles e xists as a right-handed, N-to-N-terminal beta(6.3) helical dimer [Lomi ze, A. L., Orechov, V. Yu., and Arseniev, A. S. (1992) Bioorg. Khim. 1 8, 182-189]. In the incorporation procedure to achieve the ion channel state of gramicidin A in SDS micelles, trifluoroethanol (TFE) is used to solubilize the hydrophobic peptide before addition to the aqueous/ micelle solution. The conformational transition of gramicidin A to for m ion channels in SDS micelles, i.e., in TFE and 10% TFE/water, has be en investigated using 2D NMR and CD spectroscopy. In neat TFE, gramici din A was found to be monomeric and may possibly exist in an equilibri um of rapidly interconverting conformers of at least three different f orms believed to be left- and/or right-handed a, and beta(4.4) helices . It was found that the interconversion between these conformers was s lowed down in 55% TFE as evident by the observation of at least three different sets of d(alpha N) COSY peaks although CD gave a net spectru m similar to that in neat TFE. In 10% TFE gramicidin A spontaneously f orms a precipitate. The precipitated species were isolated and solubil ized in dioxane where gramicidin conformers undergo very slow intercon version and could be characterized by NMR. At least seven different gr amicidin A conformations were found in 10% TFE. Four of these are the same types of double helices as previously found in ethanol (i.e., a s ymmetric left-handed parallel beta(5.6) double helix, an unsymmetric l eft-handed parallel beta(5.6) double helix, a symmetric left-handed an tiparallel beta(5.6) double helix, a symmetric right-handed parallel b eta(5.6) double helix); the fifth is possibly a symmetric right-handed antiparallel beta(5.6) double helix. There is also evidence for the p resence of at least one form of monomeric species. Previous observatio n on the solvent history dependence in the ease of channel incorporati on may be explained by the presence of several different folding pathw ays to channel formation. To test this proposal, the conformation of g ramicidin A in 10% DMSO and 10% methanol was studied. In the former en vironment, the major form was a random coil with a minor population of double-stranded helices, while in the latter, NMR spectra indicate th e presence of the same double-helical conformers as found in neat meth anol.