The lipid bilayer component of biological membranes is important for the distribution, organization, and function of bilayer-spanning proteins. This regulation is due to both specific lipid-protein interactions and general bilayer-protein interactions, which modulate the energetics and kinetics of protein conformational transitions, as well as the protein distribution between different membrane compartments. The bilayer regulation of membrane protein function arises from the hydrophobic coupling between the protein's hydrophobic domains and the bilayer hydrophobic core, which causes protein conformational changes that involve the protein/bilayer boundary to perturb the adjacent bilayer. Such bilayer perturbations, or deformations, incur an energetic cost, which for a given conformational change varies as a function of the bilayer material properties (bilayer thickness, intrinsic lipid curvature, and the elastic compression and bending moduli). Protein function therefore is regulated by changes in bilayer material properties, which determine the free-energy changes caused by the protein-induced bilayer deformation. The lipid bilayer thus becomes an allosteric regulator of membrane function.

We used solid-state deuterium NMR spectroscopy and an approach involving geometric analysis of labeled alanines (GALA method) to examine the structure and orientation of a designed synthetic hydrophobic, membrane-spanning alpha-helical peptide in phosphatidylcholine (PC) bilayers. The 19-amino-acid peptide consists of an alternating leucine and alanine core, flanked by tryptophans that serve as interfacial anchors: acetyl-GWW(LA)(6)LWWA-ethanolamine (WALP19). A single deuterium-labeled alanine was introduced at different positions within the peptide. Peptides were incorporated in oriented bilayers of dilauroyl- (di-C12:0-), dimyristoyl- (di-C14:0-), or dioleoyl- (di-C18:1(c)-) phosphatidylcholine. The NMR data fit well to a WALP19 orientation characterized by a distinctly nonzero tilt, approximately 4 degrees from the membrane normal, and rapid reorientation about the membrane normal in all three lipids. Although the orientation of WALP19 varies slightly in the different lipids, hydrophobic mismatch does not seem to be the dominant factor causing the tilt. We suggest rather that the peptide itself has an inherently preferred tilted orientation, possibly related to peptide surface characteristics or the disposition of tryptophan indole anchors relative to the lipids, the peptide backbone, and the membrane/water interface. Additionally, the data allow us to define more precisely the local alanine geometry in this membrane-spanning alpha-helix.

Specific interactions of membrane proteins with the membrane interfacial region potentially define protein position with respect to the lipid environment. We investigated the proposed roles of tryptophan and lysine side chains as "anchoring" residues of transmembrane proteins. Model systems were employed, consisting of phosphatidylcholine lipids and hydrophobic ␣-helical peptides, flanked either by tryptophans or lysines. Peptides were incorporated in bilayers of different thickness, and effects on lipid structure were analyzed. Induction of nonbilayer phases and also increases in bilayer thickness were observed that could be explained by a tendency of Trp as well as Lys residues to maintain interactions with the interfacial region. However, effects of the two peptides were remarkably different, indicating affinities of Trp and Lys for different sites at the interface. Our data support a model in which the Trp side chain has a specific affinity for a well defined site near the lipid carbonyl region, while the lysine side chain prefers to be located closer to the aqueous phase, near the lipid phosphate group. The information obtained in this study may further our understanding of the architecture of transmembrane proteins and may prove useful for refining prediction methods for transmembrane segments.In biological membranes, a variety of interactions can occur between lipids and proteins that affect protein as well as lipid properties and in which both the hydrophobic membrane core and the more polar membrane interfaces can be involved (1-3). Membrane proteins are able to span the lipid bilayer through interactions of their exposed hydrophobic segments with the lipid hydrocarbon acyl chains. In general, the length of these hydrophobic segments will approximately match the membrane hydrophobic thickness. However, also a mismatch between protein hydrophobic length and membrane hydrophobic thickness may occur. Such a mismatch can have considerable influence on membrane structure and function (reviewed in Ref. 4) and may, for example, be involved in protein sorting, microdomain formation, changes in protein activity, or changes in lipid structure and organization.In contrast to the hydrophobic core of a membrane, the membrane interface presents a complex and heterogeneous chemical environment, which accounts for a relatively large proportion of the total bilayer thickness (3). Specific interactions of membrane proteins with the interfacial region of the lipids may influence many functional processes, such as for instance membrane protein assembly, topology of membrane proteins, the mode of protein insertion into the membrane, and protein anchoring to the membrane. In addition, such interactions may play a determining role in hydrophobic mismatch (4).Analyses of the structure of transmembrane proteins suggest that two types of amino acids may be of special importance for interactions of membrane proteins with the interfacial region: aromatic amino acids, in particular tryptophans, which are enriched at both ends of tr...

We have investigated the effect of several hydrophobic polypeptides on the phase behavior of diacylphosphatidylcholines with different acyl chain length. The polypeptides 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. First it was demonstrated by circular dichroism measurements that these peptides adopt an alpha-helical conformation with a transmembrane orientation in bilayers of dimyristoylphosphatidylcholine. Subsequent 31P NMR measurements showed that the peptides can affect lipid organization depending on the difference in hydrophobic length between the peptide and the lipid bilayer in the liquid-crystalline phase. When a 17 amino acid residue long peptide (WALP17) was incorporated in a 1/10 molar ratio of peptide to lipid, a bilayer was maintained in saturated phospholipids containing acyl chains of 12 and 14 C atoms, an isotropic phase was formed at 16 C atoms, and an inverted hexagonal (HII) phase at 18 and 20 C atoms. For a 19 amino acid residue long peptide (WALP19) similar changes in lipid phase behavior were observed, but at acyl chain lengths of 2 C-atoms longer. Also in several cis-unsaturated phosphatidylcholine model membranes it was found that these peptides and a shorter analog (WALP16) induce the formation of nonbilayer structures as a consequence of hydrophobic mismatch. It is proposed that this unique ability of the peptides to induce nonbilayer structures in phosphatidylcholine model membranes is due to the presence of two tryptophans at both sides of the membrane/water interface, which prevent the peptide from aggregating when the mismatch is increased. Comparison of the hydrophobic length of the bilayers with the length of the different peptides showed that it is the precise extent of mismatch that determines whether the preferred lipid organization is a bilayer, isotropic phase, or HII phase. The peptide-containing bilayer and HII phase were further characterized after sucrose density gradient centrifugation of mixtures of WALP16 and dioleoylphosphatidylcholine. 31P NMR measurements of the isolated fractions showed that a complete separation of both components was obtained. Chemical analysis of these fractions in samples with varying peptide concentration indicated that the HII phase is highly enriched in peptide (peptide/lipid molar ratio of 1/6), while the maximum solubility of the peptide in the lipid bilayer is about 1/24 (peptide/lipid, molar). A molecular model of the peptide-induced HII phase is presented that is consistent with the results obtained thus far.