A polyalanine-based peptide cannot form a stable transmembrane alpha-helix in fully hydrated phospholipid bilayers.

The conformation and amide proton exchangeability of the peptide acetyl-K(2)-A(24)-K(2)-amide (A(24)) and its interaction with phosphatidylcholine bilayers were examined by a variety of physical techniques. When dissolved in or cast from methanol as a dried film, A(24) is predominantly alpha-helical. In aqueous media, however, A(24) exists primarily as a mixture of helical (though not necessarily alpha-helical) and random coiled structures, both of which allow rapid H-D exchange of all amide protons. When incorporated into phospholipids in the absence of water, A(24) also exists primarily as a transmembrane alpha-helix. However, upon hydration of that system, rapid exchange of all amide protons also occurs along with a marked change in the amide I absorption band of the peptide. Also, when dispersed with phosphatidylcholine in aqueous media, the conformation and thermal stability of A(24) are not significantly altered by the presence of the phospholipid or by its gel/liquid-crystalline phase transition. Differential scanning calorimetric and electron spin resonance spectroscopic studies indicate that A(24) has relatively minor effects on the thermodynamic properties of the lipid hydrocarbon chain-melting phase transition, that it does not abolish the lipid pretransition, and that its presence has no significant effect on the orientational order or rates of motion of the phospholipid hydrocarbon chains. We therefore conclude that A(24) has sufficient alpha-helical propensity, but insufficient hydrophobicity, to maintain a stable transmembrane association with phospholipid bilayers in the presence of water. Instead, it exists primarily as a dynamic mixture of helices and other conformers and resides mostly in the aqueous phase where it interacts weakly with the bilayer surface or with the polar/apolar interfacial region of phosphatidylcholine bilayers. Thus, polyalanine-based peptides are not good models for the transmembrane alpha-helical segments of natural membrane proteins.