Folding of β-sheet membrane proteins: A hydrophobic hexapeptide model

Beta-sheets, in the form of the β-barrel folding motif, are found in several constitutive membrane proteins (porins) and in several microbial toxins that assemble on membranes to form oligomeric transmembrane channels. We report here a first step towards understanding the principles of β-sheet formation in membranes. In particular, we describe the properties of a simple hydrophobic hexapeptide, acetyl-Trp-leu₅ (AcWL₅), that assembles cooperatively into β-sheet aggregates upon partitioning into lipid bilayer membranes from the aqueous phase where the peptide is strictly monomeric and random coil. The aggregates, containing 10 to 20 monomers, undergo a relatively sharp and reversible thermal unfolding at ~ 60°C. No pores are formed by the aggregates, but they do induce graded leakage of vesicle contents at very high peptide to lipid ratios. Because β-sheet structure is not observed when the peptide is dissolved in n-octanol, trifluoroethanol or sodium dodecyl sulfate micelles, aggregation into β-sheets appears to be an exclusive property of the peptide in the bilayer membrane interface. This is an expected consequence of the hypothesis that a reduction in the free energy of partitioning of peptide bonds caused by hydrogen bonding drives secondary structure formation in membrane interfaces. But, other features of interfacial partitioning, such as side-chain interactions and reduction of dimensionality, must also contribute. We estimate from our partitioning data that the free energy reduction per residue for aggregation is about 0.5 kcal mol^-1. Although modest, its aggregate effect on the free energy of assembling β-sheet proteins can be huge. This surprising finding, that a simple hydrophobic hexapeptide readily assembles into oligomeric β-sheets in membranes, reveals the potent ability of membranes to promote secondary structure in peptides, and shows that the formation of β-sheets in membranes is more facile than expected. Furthermore, it provides a basis for understanding the observation that membranes promote self-association of β-amyloid peptides. AcWL₅, and related peptides thus provide a good starting point for designing peptide models for exploring the principles of β-sheet formation in membranes.