Probing the lipid-protein interface using model transmembrane peptides with a covalently linked acyl chain.

The aim of this study was to gain insight into how interactions between proteins and lipids in membranes are sensed at the protein-lipid interface. As a probe to analyze this interface, we used deuterium-labeled acyl chains that were covalently linked to a model transmembrane peptide. First, a perdeuterated palmitoyl chain was coupled to the Trp-flanked peptide WALP23 (Ac-CGWW(LA)(8)LWWA-NH(2)), and the deuterium NMR spectrum was analyzed in di-C18:1-phosphatidylcholine (PC) bilayers. We found that the chain order of this peptide-linked chain is rather similar to that of a noncovalently coupled perdeuterated palmitoyl chain, except that it exhibits a slightly lower order. Similar results were obtained when site-specific deuterium labels were used and when the palmitoyl chain was attached to the more-hydrophobic model peptide WLP23 (Ac-CGWWL(17)WWA-NH(2)) or to the Lys-flanked peptide KALP23 (Ac-CGKK(LA)(8)LKKA-NH(2)). The experiments showed that the order of both the peptide-linked chains and the noncovalently coupled palmitoyl chains in the phospholipid bilayer increases in the order KALP23 < WALP23 < WLP23. Furthermore, changes in the bulk lipid bilayer thickness caused by varying the lipid composition from di-C14:1-PC to di-C18:1-PC or by including cholesterol were sensed rather similarly by the covalently coupled chain and the noncovalently coupled palmitoyl chains. The results indicate that the properties of lipids adjacent to transmembrane peptides mostly reflect the properties of the surrounding lipid bilayer, and hence that (at least for the single-span model peptides used in this study) annular lipids do not play a highly specific role in protein-lipid interactions.

A synergistic effect between cholesterol and tryptophan-flanked transmembrane helices modulates membrane curvature.

The aim of this study was to gain insight into the structural consequences of hydrophobic mismatch for membrane proteins in lipid bilayers that contain cholesterol. For this purpose, tryptophan-flanked peptides, designed to mimic transmembrane segments of membrane proteins, were incorporated in model membranes of unsaturated phosphatidylcholine bilayers of varying thickness and containing varying amounts of cholesterol. Analysis of the lipid organization by (31)P NMR and cryo-TEM demonstrated the formation of an isotropic phase, most likely representing a cubic phase, which occurred exclusively in mixtures containing lipids with relatively long acyl chains. Formation of this phase was inhibited by incorporation of lysophosphatidylcholine. These results indicate that the isotropic phase is formed as a consequence of negative hydrophobic mismatch and that its formation is related to a negative membrane curvature. When either peptide or cholesterol was omitted from the mixture, isotropic-phase formation did not occur, not even when the concentrations of these compounds were significantly increased. This suggests that formation of the isotropic phase is the result of a synergistic effect between the peptides and cholesterol. Interestingly, isotropic-phase formation was not observed when the tryptophans in the peptide were replaced by either lysines or histidines. We propose a model for the mechanism of this synergistic effect, in which its dependence on the flanking residues is explained by preferential interactions between cholesterol and tryptophan residues.

Sphingomyelin is much more effective than saturated phosphatidylcholine in excluding unsaturated phosphatidylcholine from domains formed with cholesterol.

In this study, we compared domain formation in raft-like mixtures of cholesterol and dioleoylphosphatidylcholine (DOPC) with either sphingomyelin (SM) or dipalmitoylphosphatidylcholine (DPPC). Using 2H nuclear magnetic resonance, we studied the properties of the lipid enriched in the fluid phase, DOPC. We found that acyl chain 2H-labeled DOPC is much less ordered in SM-containing mixtures than in those containing DPPC, suggesting that DOPC in the SM-containing mixture senses a lower concentration of cholesterol in its direct environment. Atomic force microscopy experiments demonstrated large differences in the size and shape of domains in the different mixtures. We propose that these various differences are a consequence of the preferential interaction of cholesterol for sphingolipids over glycerophospholipids.

Influence of hydrophobic mismatch and palmitoylation on the association of transmembrane alpha-helical peptides with detergent-resistant membranes.

The aim of this study was to gain insight into the mechanism through which transmembrane proteins are targeted to liquid ordered (L(o)) phase domains or rafts. This was investigated by analyzing the Triton X-100 resistance of designed transmembrane peptides in model membranes of 1,2-dioleoyl-sn-glycero-3-phosphocholine, sphingomyelin and cholesterol (1/1/1, molar ratio), which contain both L(o) phase domains and fluid bilayers. By using peptides with one or two palmitate chains covalently linked to their N-terminus or with variable hydrophobic lengths, the roles of protein palmitoylation and of mismatch between the transmembrane segment of the protein and the bilayer thickness, respectively, were investigated. The results show that neither hydrophobic matching nor palmitoylation is sufficient for partitioning of peptides into L(o) phase domains. It is concluded that the L(o) phase itself, due to the tight packing of the lipids, constitutes an unfavorable environment for accommodation of protein transmembrane segments.