Conformations of proline residues in membrane environments

Although noted as hydrophilic residues with helix‐breaking potential, proline residues are observed in putatively α‐helical transmembrane (TM) segments of many channel‐forming integral membrane proteins. In addition to the recognized property of X‐Pro peptide bonds (where X = any amino acid) to occur in cis as well as trans isomeric states, the tertiary amide character of the X‐Pro bond confers increased propensity for involvement of its carbonyl group in specific H‐bonded structures (e.g., β‐ and γ‐turns) and/or liganding interactions with positively charged species. To examine this latter situation in further detail, we identified Leu‐Pro‐Phe as a consensus sequence triad based on actual occurrences of intramembranous Pro residues in transport protein TM segments. Accordingly, we have undertaken the synthesis of hydrophobic peptides with potential membrane affinity, of which t ‐butyloxycarbonyl‐ L ‐Ala‐ L ‐Ala‐ L ‐Leu‐ L ‐Pro‐ L ‐Phe‐OH ( t ‐Boc‐AAALPF‐OH) is an initial compound. Partitioning of this peptide into model membrane environments composed of lipid micelles induces specific conformations(s) for the membrane‐bound hexapeptide, as monitored by 75‐MHz 13 C‐nmr spectral behavior of 13 C‐enriched Leu and Pro carbonyl carbons, and by 300‐MHz 1 H‐nmr spectra of peptide α, β, and aromatic protons. Data are interpreted in terms of an intramolecularly H‐bonded inverse γ‐turn conformation in the membrane environment involving the Leu‐Pro‐Phe triad. The inherent structureal instability of a Pro‐containing segment in a TM helix due to the multiplicity of possible local conformations is discussed as a functional aspect of membrane‐buried prolines in transport proteins.