Response of GWALP Transmembrane Peptides to Incorporation of Buried Histidine Residues

Abstract

To investigate histidine residue replacements in lipid bilayer membranes, we have employed GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide) as a favorable host peptide framework. 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 of GWALP23-H12 reveal a marked difference in peptide behavior between acidic and neutral pH conditions. At neutral pH, GWALP23-H12 and GWALP23-H13 exhibit well-defined tilted transmembrane orientations in both DOPC and DLPC bilayer membranes. Under acidic conditions GWALP23-H12 and GWALP23-H13 are highly dynamic and exhibit multiple states. Indeed, the multi-state behavior of GWALP23-H12 and GWALP23-H13 between pH 1.5 and pH 3 resembles closely that of GWALP23-R12 at neutral pH (J. Am. Chem. Soc. 132, 5803). The dramatic change in the behavior of each peptide suggests a pKa value of less than 3 to yield the neutral His imidazole side chain when buried in a lipid bilayer. Chemical exchange of the C2 imidazole proton for deuterium introduces a probe which potentially allows for direct observation of the His ring by solid-state 2H NMR over a range of conditions. Multiple His residues further alter the peptide properties, as GWALP23-H12,13 appears to aggregate in DLPC and DOPC bilayers over a range of pH conditions. Similar patterns are observed with GWALP23-H12,14; yet the 2H quadrupolar splittings for the β = 90° and β = 0° membrane orientations suggest different helix dynamics. Further aspects of the pH dependence of transmembrane helices having one or two histidine residues are under investigation.