Influence of Cholesterol on Single Arginine-Containing Transmembrane Helical Peptides

Abstract

Polar amino acids within the helical core of a transmembrane sequence, although sparse, are essential to the structure and function of many membrane proteins. In order to examine polar residues in lipid bilayer membranes, it is useful to employ a peptide framework such as GWALP23 (acetyl-GGALWLALALALALALALWLAGA-amide). GWALP23 and related model peptides fold into helices and adopt defined tilted orientations that can be observed in bilayer membranes by means of solid-state NMR spectra from particular labels. For example, it is convenient to include specific 2H-labeled alanine residues within the helical core of the peptide. GWALP23 is a favorable host peptide for single residue replacements due to the limited dynamic averaging of NMR observables such as the deuterium quadrupolar splittings of the alanine side chains. The goal of this project is to study the influence of arginine residues and pH on helix behavior in cholesterol-containing bilayers. GWALP23-R14 was incorporated into DOPC bilayers with varying amounts of cholesterol (0-20%). Although 10% cholesterol has little effect on the orientation of GWALP23-R14 in DOPC bilayers, solid-state 2H NMR spectra reveal a marked difference in peptide behavior when 20% cholesterol is present in the bilayers. Multiple peaks in the 2H NMR spectra of GWALP23-R14 (Ala d4: 1150% and 13100%) in DOPC bilayers with 20% cholesterol at high pH suggest a multi-state behavior of the peptide. Changes in the magnitudes of the 2H quadrupolar splittings furthermore suggest that 20% cholesterol may alter the helix tilt even at lower pH. The results reveal a sensitivity of peptide helix properties to cholesterol.