A polyalanine-based peptide cannot form a stable transmembrane alpha-helixin 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 bilay ers were examined by a variety of physical techniques. When dissolved in or cast from methanol as a dried film, A24 is predominantly a-helical. In aqu eous media, however, A24 exists primarily as a mixture of helical (though n ot necessarily a-helical) and random coiled structures, both of which allow rapid H-D exchange of all amide protons. When incorporated into phospholip ids in the absence of water, A24 also exists primarily as a transmembrane a -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 b and of the peptide. Also, when dispersed with phosphatidylcholine in aqueou s media. the conformation and thermal stability of A(24) are not significan tly altered by the presence of the phospholipid or by its gel/liquid-crysta lline phase transition. Differential scanning calorimetric and electron spi n resonance spectroscopic studies indicate that A24 has relatively minor ef fects on the thermodynamic properties of the lipid hydrocarbon chain-meltin g phase transition, that it does not abolish the lipid pretransition, and t hat its presence has no significant effect on the orientational order or ra tes of motion of the phospholipid hydrocarbon chains. We therefore conclude that A24 has sufficient a-helical propensity, but insufficient hydrophobic ity, to maintain a stable transmembrane association with phospholipid bilay ers in the presence of water. Instead, it exists primarily as a dynamic mix ture of helices and other conformers and resides mostly in the aqueous phas e where it interacts weakly with the bilayer surface or with the polar/apol ar interfacial region of phosphatidylcholine bilayers. Thus, polyalanine-ba sed peptides are not good models for the transmembrane alpha -helical segme nts of natural membrane proteins.