Phase separation and hexagonal H II phase formation by gramicidins A, B and C in dioleoylphosphatidylcholine model membranes. A study on the role of the tryptophan residues

Abstract

The role of the tryptophan-residues in gramicidin-induced H II phase formation was investigated in dioleoylphosphatidylcholine (DOPC) model membranes. 31P-NMR and small angle X-ray diffraction measurements showed, that gramicidin A and C (in which tryptophan-11 is replaced by tyrosine) induce a similar extent of H II phase formation, whereas for gramicidin B and synthetic analogs in which one tryptophan, either at position 9 or 11 is replaced by phenylalanine, a dramatic decrease of the H II phase inducing activity can be observed. Modification of all four tryptophans by means of formylation of the indole NH group leads to a complete block of H II phase formation. Sucrose density centrifugation experiments on the various peptide/lipid samples showed a quantitative incorporation of the peptide into the lipid. For all samples in a 1 10 molar ratio of peptide to lipid distinct bands were found, indicative of a phase separation. For the gramicidin A′/DOPC mixture these bands were analyzed and the macroscopic organization was determined by 31P-NMR and small-angle X-ray diffraction. The results demonstrate that a quantitative phase separation had occurred between a lamellar phase with a gramicidin/lipid ratio of 1 15 and a hexagonal H II phase, which is highly enriched in gramicidin. A study on the hydration properties of tryptophan- N-formylated gramicidin in mixtures with DOPC showed that this analog has a similar dehydrating effect on the lipid headgroup as the unmodified gramicidin. In addition both the hydration study and sucrose density centrifugation experiments showed that, like gramicidin also its analogs have a tendency to aggregate, but with differences in aggregation behaviour which seemed related to their H II phase inducing activity. It is proposed (i) that the main driving force for H II phase formation is the tendency of gramicidin molecules to self-associate and organize into tubular structures such as found in the H II phase and (ii) that whether gramicidin (analogs) form these or other types of aggregates depends on their tertiary structure, which is determined by intra- as well as intermolecular aromatic-aromatic stacking interactions.