CONFORMATIONAL TRANSITIONS OF GRAMICIDIN A IN PHOSPHOLIPID MODEL MEMBRANES - A HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY ASSESSMENT

We have investigated the conformation of gramicidin A reconstituted in different phospholipid environments, small unilamellar vesicles, extensive bilayers, and micelles, by exploiting a recently proposed experimental approach based on high-performance liquid chromatography [Bano et al. (1988) J. Chromatogr. 458, 105; Bano et al. (1988) FEBS Lett. 250, 67]. The method allows the separation of conformational species of the peptide, namely, antiparallel double-stranded (APDS) dimers and beta-6.3-helical monomers, and quantitation of their proportions in the lipid environment. Various experimental parameters (e.g., nature of organic solvent, time of incubation in organic solvent, lipid-to-peptide mole ratio, time of sonication, and temperature) commonly involved in sample preparation protocols have been analyzed independently. The results show how the peptide conformation in model membranes is exquisitely dictated by the particular nature of the reconstitution protocol. In addition, we have elucidated the nature of the slow conformational transition of gramicidin toward the channel configuration that takes place upon incubation of the model membranes. This transition has been characterized as a temperature-dependent conversion from APDS dimeric to beta-6.3-helical monomeric forms. Analysis of kinetic data permits an accurate calculation of the rate constant for this process at different temperatures in phospholipid vesicles and micelles. Finally, an explanation is proposed for the laboratory-to-laboratory variation in the observed spectral patterns of inserted gramicidin. Evidence is presented that a given circular dichroism spectrum can be attributed mainly to the contributions of two well-defined conformational species, APDS dimers and beta-6.3-helical monomers, coexisting in the lipid environment in a proportion which is in turn determined by the specific protocol followed for sample preparation.