Charged and Aromatic Anchoring Amino Acids Affect the Orientation of Transmembrane Peptides: A Deuterium NMR Study

Abstract

In integral proteins, the membrane-spanning segments are often flanked by aromatic and/or charged amino acids, which serve as anchoring residues, promoting protein-lipid interactions. These residues have specific polarity preferences, which are reflected in their typical immersion depths. Namely, tryptophan (W) is localized primarily to the carbonyl region of lipid acyl chains, while charged lysine (K) and arginine (R) prefer more polar regions. Although such patterns are widely recognized, little is known regarding the contributions of these residues for the orientations of transmembrane segments. Solid-state NMR spectroscopy offers a way to approach this problem. Peptides of the “WALP” family, GWW(LA)nLWWA, have served as useful models. Membrane-spanning proteins, nevertheless, typically contain more diverse sets of anchoring amino acids, which may act together to influence molecular geometry and orientation. To address these issues, we have designed the X2,22W5,19ALP23 peptides (acetyl-GXALW5(LA)6LW19LAXA-[ethanol]amide) to encompass two different anchor residues. In these peptides the inner anchor is kept constant (W), while the outer “anchor” (separated by a short Leu-Ala spacer) is varied to either W, K, R or the control residue G. Solid-state 2H NMR spectra were recorded for peptides having Ala CD3 groups in aligned bilayers of DLPC, DMPC and DOPC. The “Geometric Analysis of Labeled Alanines” provided a means to deduce the apparent average peptide orientations. Introduction of charged anchors (X = K or R) resulted in a 2-5° increase in the apparent tilt angle compared to X = G. Conversely, for X = W, the apparent tilt angle decreases 2-9°, accompanied by a significant change in the tilt direction.