Helix Fraying May Stabilize Transmembrane Alpha Helices

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. 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. To this end, we have substituted positions 4 and 5 of GWALP23 with either two Phe residues or two Ala residues to generate F4,5GWALP23 (acetyl-GGAF4F5(LA)6LW19LAGA-ethanolamide) or A4,5GWALP23 (acetyl-GGAA4A5(LA)6LW19LAGA-ethanolamide). By incorporating specific 2H-Ala labels at A3 and A21, as well as within the (LA)6L core, we are able to compare the influence of interfacial residues on the integrity of the core helix and the extent of unwinding of the helix ends. Solid state 2H NMR spectra of macroscopically aligned bilayer samples indicate a well oriented, tilted core helix for the (LA)6L sequence of A4,5GWALP23 as well as F4,5GWALP23 in DLPC, DMPC and DOPC lipid bilayers. Furthermore, the spectra from deuterium labels on alanines 3 and 21 show substantial unwinding at the terminals for both F4,5GWALP23 and A4,5GWALP23. Further studies will address (a) the precise point of N-terminal unwinding of A4,5GWALP23 by use of 2H labels at A4 and A5 and (b) the possibility of unwinding of Y4,5GWALP23 and G4,5GWALP23 helices. The fraying of helix ends may be vital for the stability of the transmembrane helix orientation in lipid-bilayer membranes.