Huibert Tournois

Interfacial properties of gramicidin and gramicidin-lipid mixtures measured with static and dynamic monolayer techniques

Gramicidin films at the air/water interface are shown to exhibit a phase transition at 225 A2/molecule which might be caused by either cluster formation, reorientation of molecules, conformational changes or multilayer formation. It is further shown that coupling of a charged group on either NH2- or COOH-terminus or elongation of the peptide by two amino acids, only slightly affects the surface area characteristics whereas modification of the tryptophans or even replacement of a single tryptophan by phenylalanine leads to drastic alterations in the surface-area characteristics and a (partial) loss of the phase transition demonstrating that the tryptophans play an important role in the interfacial behavior of gramicidin. The lack of a solvent history effect on the interfacial behavior indicates a rapid conformational interconversion of the peptide at the air/water interface. Gramicidin in mixtures with dioleoylphosphatidylcholine and lysopalmitoylphosphatidylcholine shows a condensing effect whereas gramicidin shows ideal mixing with dioleoylphosphatidylethanolamine. The condensing effect most likely is related to the aggregational state of the peptides which is different in phosphatidylcholines and phosphatidylethanolamines.

Influence of cholesterol on gramicidin-induced HII phase formation in phosphatidylcholine model membranes

The influence of cholesterol incorporation on gramicidin-induced hexagonal HII phase formation in different phosphatidylcholine model systems was investigated by 31P- and 2H-NMR, small-angle X-ray diffraction and differential scanning calorimetry. In liquid-crystalline distearoylphosphatidylcholine systems cholesterol inhibits gramicidin-induced HII phase formation. In dioleoylphosphatidylcholine the opposite effect is observed. Cholesterol appears to preferentially interact with gramicidin under liquid-crystalline conditions in both systems. Two phenomena that had been reported for gramicidin-treated erythrocyte membranes and derived liposomes (Tournois, H., Leunissen-Bijvelt, J., Haest, C.W.M., De Gier, J. and De Kruijff, B. (1987) Biochemistry, 26, 6613-6621) could also be observed in more simple dioleoylphosphatidylcholine-gramicidin-cholesterol systems. These are (i) an increase in tube diameter in the gramicidin-induced HII phase with increasing temperature, which is ascribed to the presence of cholesterol in this phase, and (ii) the loss of the hexagonal HII phase related 31P-NMR line shape at lower temperatures despite the presence of this phase as demonstrated with X-ray diffraction. This latter phenomenon appears to be due to restrictions in the rate of lateral diffusion of the phospholipids around the HII tubes due to the presence of gramicidin.

Gramicidin-induced hexagonal HII phase formation in negatively charged phospholipids and the effect of N- and C-terminal modification of gramicidin on its interaction with zwitterionic phospholipids

The effect of gramicidin on macroscopic structure of the negatively charged membrane phospholipids cardiolipin, dioleoylphosphatidylglycerol and dioleoylphosphatidylserine in aqueous dispersions was investigated and compared with the effect of gramicidin on dioleoylphosphatidylcholine. It was shown by small-angle X-ray diffraction, 31P nuclear magnetic resonance and freeze-fracture electron microscopy that in all these lipid systems gramicidin is able to induce the formation of a hexagonal HII phase. 31P-NMR measurements indicated that the extent of HII phase formation in the various lipids ranged from about 40% to 60% upon gramicidin incorporation in a molar ratio of peptide to lipid of 1 : 10. Next, the following charged analogues of gramicidin were prepared: desformylgramicidin, N-succinylgramicidin and O-succinylgramicidin. The synthesis was verified with 13C-NMR and the effect of these analogues on lipid structure was investigated. It was shown that, as with gramicidin itself, the analogues induce HII phase formation in dioleoylphosphatidylcholine, lower and broaden the bilayer-to-HII phase transition in dielaidoylphosphatidylethanolamine and form lamellar structures upon codispersion with palmitoyllysophosphatidylcholine. Differential scanning calorimetry measurements indicated that, again like gramicidin, in phosphatidylethanolamine the energy content of the gel-to-liquid-crystalline phase transition is not affected by incorporation of the analogues, whereas in phosphatidylcholine a reduction of the transition enthalpy is found. These observations were explained in terms of a similar tendency to self-associate for gramicidin and its charged analogues. The results are discussed in the light of the various factors which have been suggested to be of importance for the modulation of lipid structure by gramicidin.

Polymorphic phospholipid phase transitions as tools to understand peptide-lipid interactions

The effect of peptides on bilayer----non-bilayer phase transitions can be used as a tool to investigate the molecular aspects of peptide-lipid interactions. In this contribution the action on membranes of the peptide antibiotic gramicidin A and the bee venom component melittin are compared. Although the known structures and locations of these peptides upon membrane binding are very different, their actions on membranes show striking parallels. A general model is proposed that explains the seemingly complex peptide-lipid interactions by making use of simple concepts.

Relationship between gramacidin conformation dependent induction of phospholipid transbilayer movement and hexagonal HII phase formation in erythrocyte membranes

Addition of gramicidin in sufficient concentration from dimethylsulfoxide or trifluoroethanol to isolated erythrocyte membranes induces hexagonal HII phase formation for the phospholipids. In contrast, addition from ethanol does not change the overall bilayer organization despite a similar extent of peptide incorporation. The same solvent dependence is observed for the enhancement of transbilayer reorientation of lysophospholipids and unspecific leak formation in intact erythrocytes at lower gramicidin concentrations. These results indicate that the (beta 6.3) conformation of the peptide is essential for all three membrane perturbing effects.

Influence of Gramicidin on Lipid Organization and Dynamics in Membranes

Gramicidin is a potent modulator of long-range membrane lipid organization and therefore a useful model to understand aspects of lipid polymorphism in relation to the structure and function of biological membranes. In this article we will review the peptide’s lipid structure-modulating activity in relation to recent studies on the lipid flip-flop enhancing ability of gramicidin in erythrocyte membranes. Special emphasis will be placed on the essential role of the conformation of gramicidin and its aggregational behavior in these events. The relationships between the other known properties of gramicidin, e.g., channel formation and involvement of gene regulation, are indicated. It is suggested that the unique concentration of the four tryptophans towards the C-terminus is the structural link between these different functional properties of the peptide.

Functional and Structural Aspects of Gramicidin-Lipid Interactions

Lipids in isolated form show polymorphism. Depending on the type of lipid and experimental conditions, different macromolecular aggregate structures are formed. for membrane lipids hydrated in excess water, under physiological conditions of ionic strength, pH and temperature, three major liquid-crystalline phases are observed. These are: the lamellar phase, the inverted (type II) hexagonal phase (Fig. 1), and the normal (type I) micellar phase. The shape-structure concept of lipid polymorphism as originally proposed by Israelachvili et al. (1976) and adapted by Cullis and De Kruijff (1979) explains remarkably well most of the current data on lipid polymorphism (Fig. 2). If the surface area in the head group region matches that of the hydrocarbon region (such as in phosphatidylcholines) the molecule has an overall cylindrical shape and prefers to organize in extended bilayers. When the head group area exceeds the hydrocarbon area, as for instance in case of detergent-like lipids such as lysophosphatidylcholines, the molecule on average is cone shaped and prefers an organization with a convex (type I) surface curvature such as found in micelles or in the related normal (type I) hexagonal phase occurring for these lipids at low water content. Phosphatidylethanolamines are key representatives of the opposite case of an excess hydrocarbon area. This results in the concave (type II) surface curvature as found in the HII phase. Head group hydration and inter- and intramolecular interactions are important determinants for the shape of the lipid molecules in the shape-structure concept. The strictiy regulated (Goldfine et al., 1987; Wieslander et al., 1981) presence of large amounts of type II lipids (lipids which in hydration prefer to organize in type II structures) and their abundance in virtually all biomembranes suggests important structural and functional roles for these types of lipids.