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.
Transmembrane helices dominate the landscape for many membrane proteins. Often flanked by interfacial aromatic residues, these transmembrane helices also contain loops and inter-helix segments, which could help in stabilizing a transmembrane orientation. Using 2 H-NMR spectroscopy to monitor bilayer incorporated model GWALP23 family peptides, we address systematically the issue of helix fraying in relation to the dynamics and orientation of closely similar individual transmembrane helices. Adjacent to a core transmembrane helix, we inserted aromatic (Phe, Trp, Tyr, His) or non-aromatic residues (Ala, Gly) into positions 4 and 5, to examine the side-chain dependency of the transmembrane orientation, dynamics and helix integrity (extent and location of unraveling). Incorporation of 2 H-alanine labels enables one to assess the helicity of the core sequence and the peptide termini. For most of the helices, we observed substantial unwinding involving at least 3 residues at both ends. For the unique case of histidine at positions 4 and 5, an extended N-terminal unwinding was observed up to residue 7. For further investigation regarding the onset of fraying, we employed A 4,5 GWALP23 with 2 H labels at residues 4 and 5 and found that the number of terminal residues involved in the unwinding depends on bilayer thicknesses and helps to govern the helix dynamics. The combined results enable us to compare and contrast the extent of fraying for each related helix, as reflected by the deviation of experimental 2 H quadrupolar splitting magnitudes of juxta-terminal alanines A3 and A21 from those represented by an ideal helix geometry.
Membrane proteins are essential for many cell processes yet are more difficult to investigate than soluble proteins. Charged residues often contribute significantly to membrane protein function. Model peptides such as GWALP23 (acetyl‐GGALW5LAL8LALALAL16ALW19LAGA‐amide) can be used to characterize the influence of specific residues on transmembrane protein domains. We have substituted R8 and R16 in GWALP23 in place of L8 and L16, equidistant from the peptide center, and incorporated specific 2H‐labeled alanine residues within the central sequence for detection by solid‐state 2H NMR spectroscopy. The resulting pattern of [2H]Ala quadrupolar splitting (Δνq) magnitudes indicates the core helix for R8,16GWALP23 is significantly tilted to give a similar transmembrane orientation in thinner bilayers with either saturated C12:0 or C14:0 acyl chains (1,2‐dilauroyl‐sn‐glycero‐3‐phosphocholine (DLPC) or 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC)) or unsaturated C16:1 Δ9 cis acyl chains. In bilayers of 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC; C18:1 Δ9 cis) multiple orientations are indicated, whereas in longer, unsaturated 1,2‐dieicosenoyl‐sn‐glycero‐3‐phosphocholine (DEiPC; C20:1 Δ11 cis) bilayers, the R8,16GWALP23 helix adopts primarily a surface orientation. The inclusion of 10–20 mol % cholesterol in DOPC bilayers drives more of the R8,16GWALP23 helix population to the membrane surface, thereby allowing both charged arginines access to the interfacial lipid head groups. The results suggest that hydrophobic thickness and cholesterol content are more important than lipid saturation for the arginine peptide dynamics and helix orientation in lipid membranes.
Membrane proteins are vital for biological function and are complex to study. Even in model peptide-lipid systems, the combined influence or interaction of pairs of chemical groups still is not well understood. Disordered proteins, whether in solution or near lipid membranes, are an emerging paradigm for the initiation and control of biological function. The disorder can involve molecular orientation as well as molecular folding. This paper reports an astonishing induction of disorder when one Glu residue is introduced into a highly stable 23-residue transmembrane helix. The parent helix is anchored by a single Arg residue, tilted at a well-defined angle with respect to the DOPC bilayer normal and undergoes rapid cone precession. When Glu is introduced two residues away from Arg, with 200° (or 160°) radial separation, the helix properties change radically to exhibit a multiplicity of three or more disordered states. The helix characteristics have been monitored by deuterium (2H) NMR spectroscopy as functions of the pH and lipid bilayer composition. The disordered multistate behavior of the (Glu, Arg)-containing helix varies with the lipid bilayer thickness and pH. The results highlight a fundamental induction of protein multistate properties by a single Glu residue in a lipid membrane environment.
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