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Helical Distortion in Tryptophan- and Lysine-Anchored Membrane-Spanning -Helices as a Function of Hydrophobic Mismatch: A Solid-State Deuterium NMR Investigation Using the Geometric Analysis of Labeled Alanines Method

Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701

Correspondence: Address reprint requests to Roger E. Koeppe 2nd, Tel.: 479-575-4601; Fax: 479-575-4049; E-mail: rk2{at}uark.edu.

We used solid-state deuterium NMR spectroscopy and geometric analysis of labeled alanines to investigate the structure and orientation of a designed synthetic hydrophobic, membrane-spanning -helical peptide that is anchored within phosphatidylcholine (PC) bilayers using both Trp and Lys side chains near the membrane/water interface. The 23-amino-acid peptide consists of an alternating Leu/Ala core sequence that is expected to be -helical, flanked by aromatic and then cationic anchors at both ends of the peptide: acetyl-GKALW(LA)₆LWLAKA-amide (KWALP23). The geometric analysis of labeled alanines method was elaborated to permit the incorporation and assignment of multiple alanine labels within a single synthetic peptide. Peptides were incorporated into oriented bilayers of dilauroyl- (di-C12:0-), dimyristoyl- (di-C14:0-), or dioleoyl- (di-C18:1c-) PC. In the C12:0 and C14:0 lipids, the ²H-NMR quadrupolar splittings for the set of six core alanines could not be fit to a canonical undistorted -helix. Rather, we found that a model containing a helical distortion, such as a localized discontinuity or "kink" near the peptide and bilayer center, could fit the data for KWALP23 in these shorter lipids. The suggestion of helix distortion was confirmed by ²H-NMR spectra for KWALP23 in which Leu⁸ was changed to deuterated Ala⁸. Further analysis involving reexamination of earlier data led to a similar conclusion that acetyl-GWW(LA)₈LWWA-amide (WALP23) is distorted in dilauroyl-PC, allowing significant improvement in the fitting of the ²H-NMR results. In contrast, WALP23 and KWALP23 are well represented as undistorted -helices in dioleoyl-PC, suggesting that the distortion could be a response to hydrophobic mismatch between peptide and lipids.