Influence of High pH and Cholesterol on Single Arginine-Containing Transmembrane Peptide Helices

Abstract

An essential component of mammalian cells, cholesterol exerts significant influence on the physical properties of the cell membrane and in turn its constituents, including membrane proteins. Although sparse, polar amino acid residues are highly conserved in membrane proteins and play pivotal roles in determining specific structural and functional properties. To improve our understanding of particular polar residues in the membrane environment, we have examined two specific “guest” Arg residues within a well-defined and deuterium-labeled “host” framework provided by the transmembrane helical peptide GWALP23 (acetyl-GGALWLALALALALALALWLAGA-amide). Solid-state 2H nuclear magnetic resonance (NMR) spectra from aligned bilayer membrane samples effectively report changes in the host helix properties because of the incorporation of the guest residues. The focus of this work is two-pronged. First, GWALP23-R14 was examined over a pH range of 2–13 in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) ester- or ether-linked bilayer membranes. Our findings indicate that the Arg guanidinium side chain remains charged over this entire range, in agreement with numerous molecular dynamics simulations. Second, GWALP23-R12 and GWALP23-R14 peptides were characterized in DOPC bilayers with varying cholesterol content. Our findings suggest that 10 or 20% cholesterol content has minimal impact on the orientation of the R14 peptide. Although the NMR signals are broader and weaker in the presence of 20% cholesterol, the deuterium quadrupolar splittings for [2H]Ala residues in GWALP23-R14 change very little. Conversely, cholesterol appears to modulate the multistate behavior of GWALP23-R12 and to favor a major interfacial state for the helix, bound at the bilayer surface. These results indicate a conditional sensitivity of a complex multistate transmembrane Arg-containing peptide helix to the presence of cholesterol.

Influence of glutamic acid residues and pH on the properties of transmembrane helices

“…These titratable protein side chains, notably those of Asp, Glu, His, Lys and Arg, encounter an environment-dependent energy barrier that governs their ability to pick up or lose a proton in transitioning between charged and neutral states. Changes in the microenvironments of these buried residues, such as the bilayer thickness [6], cholesterol content [7], lipid phosphate head group identity, or presence of water molecules, therefore, may have significant influence on the charge status or preferred location of particular residues.…”

“…Notably, this pK a value is about four pH units lower than the standard value for lysine in aqueous solution. Further titration experiments with GWALP23-R14 reveal that bilayer-incorporated arginine does not titrate below pH 13, with Arg essentially preferring to exit the lipid bilayer rather than to lose a proton [7, 8, 21]. It is interesting to note that the bilayer environment, in comparison to a bulk water environment, influences the pK a of lysine much more than that of arginine.…”

Influence of Lipid Saturation, Hydrophobic Length and Cholesterol on Double‐Arginine‐Containing Helical Peptides in Bilayer Membranes

“…[5] To examinet he effect of cholesterol on the multistate behavior of GWALP23-R 8,16 in DOPC, 10 or 20 %c holesterol was included with DOPC and Figures 3 and S8, the addition of even 10 %c holesterol decreases the multistate behavior of GWALP23-R 8,16 in favor of as ingle helix, bound at the bilayer surface. [5] To examinet he effect of cholesterol on the multistate behavior of GWALP23-R 8,16 in DOPC, 10 or 20 %c holesterol was included with DOPC and Figures 3 and S8, the addition of even 10 %c holesterol decreases the multistate behavior of GWALP23-R 8,16 in favor of as ingle helix, bound at the bilayer surface.…”

“…The resulting pattern of [ 2 H]Ala quadrupolar splitting (Dn q )m agnitudesi ndicates the core helix for R 8,16 GWALP23 is significantly tilted to give as imilart ransmembrane orientation in thinner bilayersw ith 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 unsa-turatedC 16:1 D9 cis acyl chains. [1,[3][4][5]30] Previously we found that introducing as ingle arginine at position1 4i n GWALP23c aused the peptideh elix to rotate by 808 and to increase its tilt by 108 in DOPC bilayers, thus allowingt he arginine to gain access to the aqueous interface of the lipid bilayer. The inclusion of 10-20 mol %c holesterol in DOPC bilayers drives more of the R 8,16 GWALP23 helix population to the membrane surface, thereby allowing both charged arginines accesst ot he interfacial lipid head groups.T he resultss uggest that hydrophobic thickness and cholesterol content are more important than lipid saturationf or the argininep eptide dynamics and helix orientation in lipid membranes.…”

“…Experiments with designed systems can serve to identify protein-lipid interactions with single-residuer esolution. [10,12] Despite retaining ap ositivec harge in am embrane environment,u pt oat least pH 12, [5] argininei shighly conserved and plays criticalf unctional roles in many membrane proteins, including sodium channels, acetylcholine receptors, integrins and others. Whereas the ionization properties of lysine and histidine are modulated in bilayer membranes, [3,4] the guanidinium side chain of argininec arriesapositive chargei ne ssentially all biological membrane environments.…”