Examination of pH dependency and orientation differences of membrane spanning alpha helices carrying a single or pair of buried histidine residues
by Afrose, F.; Martfeld, A. N.; Greathouse, D. V.; Koeppe, R. E.
We have employed the peptide framework of GWALP23 (acetyl-GGALWLALALALALALALWLAGA-amide) to examine the orientation, dynamics and pH dependence of peptides having buried single or pairs of histidine residues. When residue L-8 is substituted to yield GWALP23-H-8, acetyl-GGALWLAH(8)ALALALALALWLAGA-amide, the deuterium NMR spectra of H-2-labeled core alanine residues reveal a helix that occupies a single transmembrane orientation in DLPC, or in DMPC at low pH, yet shows multiple states at higher pH or in bilayers of DOPC. Moreover, a single histidine at position 8 or 16 in the GWALP23 framework is sensitive to pH. Titration points are observed near pH 3.5 for the deprotonation of H-8 in lipid bilayers of DLPC or DMPC, and for H-16 in DOPC. When residues L-8 and L-16 both are substituted to yield GWALP23-H-8,H-16, the H-2 NMR spectra show, interestingly, no titration dependence from pH 2-8, yet bilayer thickness-dependent orientation differences. The helix with H-8 and H-16 is found to adopt a transmembrane orientation in thin bilayers of DLPC, a combination of transmembrane and surface orientations in DMPC, and then a complete transition to a surface bound orientation in the thicker DPoPC and DOPC lipid bilayers. In the surface orientations, alanine A7 no longer fits within the core helix. These results along with previous studies with different locations of histidine residues suggest that lipid hydrophobic thickness is a first determinant and pH a second determinant for the helical orientation, along with possible side-chain snorkeling, when the His residues are incorporated into the hydrophobic region of a lipid membrane-associated helix.