WALP Peptides Research Articles

The Effect of Peptide/Lipid Hydrophobic Mismatch on the Phase Behavior of Model Membranes Mimicking the Lipid Composition in Escherichia coli Membranes

The effect of hydrophobic peptides on the lipid phase behavior of an aqueous dispersion of dioleoylphosphatidylethanolamine and dioleoylphosphatidylglycerol (7:3 molar ratio) was studied by 31P NMR spectroscopy. The peptides (WALP n peptides, where n is the total number of amino acid residues) are designed as models for transmembrane parts of integral membrane proteins and consist of a hydrophobic sequence of alternating leucines and alanines, of variable length, that is flanked on both ends by tryptophans. The pure lipid dispersion was shown to undergo a lamellar-to-isotropic phase transition at ∼60°C. Small-angle x-ray scattering showed that at a lower water content a cubic phase belonging to the space group Pn3m is formed, suggesting also that the isotropic phase in the lipid dispersion represents a cubic liquid crystalline phase. It was found that the WALP peptides very efficiently promote formation of nonlamellar phases in this lipid system. At a peptide-to-lipid (P/L) molar ratio of 1:1000, the shortest peptide used, WALP16, lowered the lamellar-to-isotropic phase transition by ∼15°C. This effect was less for longer peptides. For all of the WALP peptides used, an increase in peptide concentration led to a further lowering of the phase transition temperature. At the highest P/L ratio (1:25) studied, WALP16 induced a reversed hexagonal liquid crystalline (H II) phase, while the longer peptides still promoted the formation of an isotropic phase. Peptides with a hydrophobic length larger than the bilayer thickness were found to be unable to inhibit formation of the isotropic phase. The results are discussed in terms of mismatch between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer and its consequences for lipid-protein interactions in membranes.

Visualization of highly ordered striated domains induced by transmembrane peptides in supported phosphatidylcholine bilayers.

We used atomic force microscopy (AFM) to study the lateral organization of transmembrane TmAW(2)(LA)(n)W(2)Etn peptides (WALP peptides) incorporated in phospholipid bilayers. These well-studied model peptides consist of a hydrophobic alanine-leucine stretch of variable length, flanked on each side by two tryptophans. They were incorporated in saturated phosphatidylcholine (PC) vesicles, which were deposited on a solid substrate via the vesicle fusion method, yielding hydrated gel-state supported bilayers. At low concentrations (1 mol %) WALP peptides induced primarily line-type depressions in the bilayer. In addition, striated lateral domains were observed, which increased in amount and size (from 25 nm up to 10 microm) upon increasing peptide concentration. At high peptide concentration (10 mol %), the bilayer consisted mainly of striated domains. The striated domains consist of line-type depressions and elevations with a repeat distance of 8 nm, which form an extremely ordered, predominantly hexagonal pattern. Overall, this pattern was independent of the length of the peptides (19-27 amino acids) and the length of the lipid acyl chains (16-18 carbon atoms). The striated domains could be pushed down reversibly by the AFM tip and are thermodynamically stable. This is the first direct visualization of alpha-helical transmembrane peptide-lipid domains in a bilayer. We propose that these striated domains consist of arrays of WALP peptides and fluidlike PC molecules, which appear as low lines. The presence of the peptides perturbs the bilayer organization, resulting in a decrease in the tilt of the lipids between the peptide arrays. These lipids therefore appear as high lines.

Influence of lipid/peptide hydrophobic mismatch on the thickness of diacylphosphatidylcholine bilayers. A 2H NMR and ESR study using designed transmembrane alpha-helical peptides and gramicidin A.

We have investigated the effect of a series of hydrophobic polypeptides (WALP peptides) on the mean hydrophobic thickness of (chain-perdeuterated) phosphatidylcholines (PCs) with different acyl chain length, using 2H NMR and ESR techniques. The WALP peptides are uncharged and consist of a sequence with variable length of alternating leucine and alanine, flanked on both sides by two tryptophans, and with the N- and C-termini blocked, e.g., FmAW2(LA)nW2AEtn. 2H NMR measurements showed that the shortest peptide with a total length of 16 amino acids (WALP16) causes an increase of 0.6 A in bilayer thickness in di-C12-PC, a smaller increase in di-C14-PC, no effect in di-C16-PC, and a decrease of 0.4 A in di-C18-PC, which was the largest decrease observed in any of the peptide/lipid systems. The longest peptide, WALP19, in di-C12-PC caused the largest increase in thickness of the series (+1.4 A), which decreased again for longer lipids toward di-C18-PC, in which no effect was noticed. WALP17 displayed an influence intermediate between that of WALP16 and WALP19. Altogether, incorporation of the WALP peptides was found to result in small but very systematic changes in bilayer thickness and area per lipid molecule, depending on the difference in hydrophobic length between the peptide and the lipid bilayer in the liquid-crystalline phase. ESR measurements with spin-labeled lipid probes confirmed this result. Because thickness is expected to be influenced most at the lipids directly adjacent to the peptides, also the maximal adaptation of these first-shell lipids was estimated. The calculation was based on the assumption that there is little or no aggregation of the WALP peptides, as was supported by ESR, and that lipid exchange is rapid on the 2H NMR time scale. It was found that even the maximal possible changes in first-shell lipid length were relatively small and represented only a partial response to mismatch. The synthetic WALP peptides are structurally related to the gramicidin channel, which was therefore used for comparison. In most lipid systems, gramicidin proved to be a stronger perturber of bilayer thickness than WALP19, although its length should approximate that of the shorter WALP16. The effects of gramicidin and WALP peptides on bilayer thickness were evaluated with respect to previous 31P NMR studies on the effects of these peptides on macroscopic lipid phase behavior. Both approaches indicate that, in addition to the effective hydrophobic length, also the physical nature of the peptide surface is a modulator of lipid order.