Detection of Helix Fraying in Transmembrane Helices with Interfacial Histidine Residues

Abstract

Transmembrane helices of integral membrane proteins often are flanked by interfacial aromatic residues that may serve as anchors to aid the stabilization of a tilted transmembrane orientation. The synthetic neutral peptide GWALP23 (acetyl-GG2ALWLALALALALALALWLAG22A-amide) with two interfacial Trp residues has proved to be surprisingly well-behaved with minimal dynamic averaging in a stable transmembrane orientation in lipid-bilayer membranes of varying thickness. To further investigate the effect of interfacial His residues, we have substituted G2 and G22 with histidine in HWALP23 (acetyl-GH2ALWLALALALALALALWLAH22A-amide). In addition, to explore the fraying or uncoiling of the ends of the peptide, we have incorporated 2H-Ala labels at positions A3 and A21 (underlined above), which are sensitive to helix integrity, outside the core region of the peptide, to compare the influence of interfacial residues on the extent of unwinding of the helix ends. Solid-state 2H NMR spectra of macroscopically aligned DOPC lipid bilayer samples and in the presence of 10% and 20% cholesterol confirmed that one or both helix ends are frayed in DOPC bilayers alone and in the presence of up to 20% cholesterol. To further understand the effects of histidine in transmembrane helices, we have also substituted W5 and W19 with histidine in GHALP23 (acetyl-GGALH5LALALALALALALH19LAGA-amide) with 2H-Ala labels at A3 and A21. “Geometric Analysis of Labeled Alanines” (GALA) shows the extent of coiling or unwinding at the terminals for HWALP23 and GHALP23. It is plausible that the helix fraying may be critical for the stability of the transmembrane helix orientation in lipid bilayer membranes.