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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 L8 is substituted to yield GWALP23-H8, acetyl-GGALWLAH8ALALALALALWLAGA-amide, the deuterium NMR spectra of 2H-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 H8 in lipid bilayers of DLPC or DMPC, and for H16 in DOPC. When residues L8 and L16 both are substituted to yield GWALP23-H8,16, the 2H NMR spectra show, interestingly, no titration dependence from pH 2–8, yet bilayer thickness-dependent orientation differences. The helix with H8 and H16 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.

In this article we review current understanding of basic principles for the folding of membrane proteins, focusing on the more abundant alpha-helical class. Membrane proteins, vital to many biological functions and implicated in numerous diseases, fold into their active conformations in the complex environment of the cell bilayer membrane. While many membrane proteins rely on the translocon and chaperone proteins to fold correctly, others can achieve their functional form in the absence of any translation apparatus or other aides. Nevertheless, the spontaneous folding process is not well understood at the molecular level. Recent findings suggest that helix fraying and loop formation may be important for overall structure, dynamics and regulation of function. Several types of membrane helices with ionizable amino acids change their topology with pH. Additionally we note that some peptides, including many that are rich in arginine, and a particular analogue of gramicidin, are able passively to translocate across cell membranes. The findings indicate that a final protein structure in a lipid-bilayer membrane is sequence-based, with lipids contributing to stability and regulation. While much progress has been made toward understanding the folding process for alpha-helical membrane proteins, it remains a work in progress. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo.