Influence of Histidine Residues on Transmembrane Helix Alignment

Abstract

To investigate His residues in lipid bilayer membranes, we have employed GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide) as a favorable host peptide. Importantly, membrane-spanning GWALP23 is quite sensitive to single-residue replacements, in part because the transmembrane helix exhibits only limited dynamic averaging of solid-state NMR observables such as the 2H quadrupolar splitting (Biophys. J. 101, 2939). We inserted His residues into position 12 and/or 13 of GWALP23 (replacing either L12 or A13) and incorporated specific 2H-Ala labels within the helical core sequence. Solid-state 2H NMR spectra reveal a marked difference between the L12H mutant and the L12H-A13H double mutant. GWALP23-H12 exhibits a well-defined tilted transmembrane orientation in both DOPC and DLPC bilayer membranes, suggesting that the bilayer-incorporated histidine side chain is neutral (deprotonated) at an experimental pH of 5-6. By contrast, GWALP23-H12,13 is highly dynamic and exhibits multiple states which are in slow exchange on the NMR timescale. Indeed, the multi-state behavior of GWALP23-H12,13 between pH 4 and pH 9 resembles closely that of GWALP23-R12 (J. Am. Chem. Soc. 132, 5803). Further aspects of the pH dependence of transmembrane helices having one or two histidines are under investigation.