Cholesterol Influence on Arginine-Containing Transmembrane Peptides

An essential component of animal cells, cholesterol exerts significant influence on the physical properties of the membrane and in turn, its constituents. One such constituent, the membrane protein, often contains polar amino acids. Although sparse, polar residues are highly conserved and play pivotal roles in determining specific structural and functional properties. To gain greater understanding of the membrane, and more broadly, cellular function, a model peptide framework termed “GWALP23” (acetyl-GGALWLALALAL12AL14ALALWLAGA-amide) is useful. The limited dynamic averaging of NMR observables such as the deuterium quadrupolar splittings of labeled alanine residues makes GWALP23 favorable for single residue replacements. Previously, GWALP23 family peptides were characterized with single Leu to Arg mutations at positions 12 and 14 in single-lipid membranes [J. Am. Chem. Soc., 132, 5803-5811, 2010]. GWALP23-R14 adopts a defined tilted orientation in DOPC bilayers, whereas GWALP23-R12 displays multi-state behavior. The goal of this research is to further characterize these peptides in cholesterol-containing bilayers. Specific deuterium-labeled alanine residues were incorporated into the R12 and R14 sequences to identify transmembrane peptide orientation by means of solid-state deuterium NMR. Both peptides were incorporated into phospholipid bilayers with varying cholesterol content (0%, 10%, or 20%). Our findings suggest that 10% or 20% cholesterol content has minimal impact on the orientation of GWALP23-R14 peptide. (Although the NMR signals are broader and weaker in the presence of 20% cholesterol, the deuterium quadrupolar splittings for 2H-Ala residues in GWALP23-R14 change little.) Conversely, cholesterol appears to reduce the multi-state behavior of GWALP23-R12, favoring a single transmembrane state for the helix. With 10% or 20% cholesterol content, the spectra exhibit defined quadrupolar splittings, suggesting that GWALP23-R12 adopts a predominant, tilted orientation in the presence of cholesterol. These results convey a conditional sensitivity of a complex multi-state peptide helix to the presence of cholesterol.