Detection of Helix Fraying in Designed Transmembrane Alpha Helices

Abstract

Transmembrane helices of integral membrane proteins often have aromatic residues flanking them. The aromatic residues favor locations within the membrane–water interface of a lipid bilayer and may serve as anchors to aid the stabilization of a tilted transmembrane orientation. To further understand the influence of Tyr, Trp or Phe residues upon the properties of helical membrane proteins (see Biochemistry 53, 3637), we have investigated the possibility of partial unwinding near the ends of selected transmembrane helices. For this purpose, we substituted positions 4 and 5 with either two Phe or two Ala residues to generate F4,5GWALP23 (acetyl-GGAF4F5(LA)6LW19LAGA-ethanolamide) or A4,5GWALP23 (acetyl-GGAA4A5(LA)6LW19LAGA-ethanolamide), respectively. By incorporating specific 2H-Ala labels at positions 3 and 21, we are able to compare the influence of interfacial Phe and Ala on the unwinding of the helix ends. Solid state NMR spectra of macroscopically aligned bilayer samples show well defined 2H-Ala quadrupolar splittings for both F4,5GWALP23 and A4,5GWALP23, suggesting that the peptide helices are well oriented in DLPC, DMPC and DOPC lipid bilayers. We are also able to estimate the helix tilt from deuterium labeling of the core alanine residues. Geometric Analysis of Labeled Alanines (GALA) then shows unwinding at the terminals for both F4,5GWALP23 and A4,5GWALP23. It is conceivable that the helix fraying may be critical for the stability of the transmembrane helix orientation in the lipid bilayer membranes.