Conformational transitions of gramicidin A in phospholipid model membranes. A high-performance liquid chromatography assessment

Abstract

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 [Bañó et al. (1988) J. Chromatogr. 458, 105; Bañó et al. (1989) 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Trapping of Different Lipase Conformers in Water-Restricted Environments

“…From a broader perspective, our trapping strategy may add to the varied repertoire of efforts reported in recent years aimed at improving our understanding of the structureactivity relationships of biomolecules (from simple peptides to large multimeric enzymes) by trapping different conformers in frozen phases or in water-restricted environments. To name a few instances: the dimer-monomer conformational transition of linear gramicidins in model bilayers and reverse micelles has been assessed in our lab by HPLC using a strategy to trap both peptide conformers (in a nonpolar mobile phase) during chromatographic elution (Ban ˜o ´et al, 1991;Salom et al, 1992Salom et al, , 1995; the trapping of both competent and incompetent conformers of triosephosphate isomerase in low-water content reverse micelles has been used in the investigation of folding and dimerization of this enzyme (Ferna ´ndez-Velasco et al, 1995); or the structure of the open-channel form of the acetylcholine receptor has been recently determined by means of an elegant strategy involving the trapping (by rapid freeze-drying) of the acetylcholine-induced structural response of the receptor and the further electron microscopy analysis of the frozen membranes [Unwin, 1995; for a recent review on the topic see Moffat and Henderson (1995)].…”

The structure of an integral membrane peptide: a deuterium NMR study of gramicidin

“…Gramicidin is known to adopt both channel and nonchannel conformations in the membrane. Fortunately, the circular dichroism (CD) signatures of each conformation are completely different and well documented (Lograsso et al, 1988;Morrow et al, 1991;Bano et al, 1991;Killian et al, 1988). We therefore employed this spectroscopic technique to confirm that the peptide had adopted the channel conformation in DLPC, using the sample preparation method described above.…”

A conformational rearrangement in gramicidin A: From a double-stranded left-handed to a single-stranded right-handed helix

“…Recently, a more detailed analysis of one of these conformers has been achieved (Pascal & Cross, 1992). However, recent studies have sometimes ignored the diversity of these structures in discussions of channel formation (Baño et al, 1989(Baño et al, , 1991.…”