Attempted “Rescue” of Glutamic Acid by Arginine in a Transmembrane Helix

Abstract

Membrane proteins are vital for biological function and are complex to study, as they must be investigated in lipid membranes. While studies of small proteins or model peptides in lipid membranes have begun to reveal some of the important lipid interactions of single residues or individual functional groups, the combined effects or interactions of even pairs of protein or peptide side chains still are not well understood. The goal of this research is to understand the combined effects of a glutamic acid and an arginine spaced two residues apart - giving 200° radial separation - within a hydrophobic transmembrane alpha-helix. To this end, we incorporated E16 and R14 the transmembrane helix of acetyl-GGALW5(LA)6LW19LAGA-amide (GWALP23) and labeled selected core alanines with deuterium for detection using 2H NMR spectroscopy. The singly substituted R14-GWALP23 shows remarkably narrow 2H NMR resonances (J Am Chem Soc 132, 5803), whereas the singly substituted E16-GWALP23 shows very broad spectra (BBA 1859, 484). Initial results for the doubly substituted R14,E16-GWALP23 show multiple populations of states for the helix in DOPC bilayers, which is very different from the effects of either residue alone. There is furthermore early indication that a major preferred helix orientation emerges when the pH is lowered from 6 to 3 in DOPC. Ongoing experiments are addressing the multi-state behavior and the pH dependence of the properties of the R14,E16-GWALP23 helix in DOPC membranes. Additional experiments will address the behavior of this helix in DLPC, DMPC and DEiPC bilayer membranes.