Varied Approaches to the Ionization Behavior of Specific Glu Residues that Face the Lipids in Transmembrane Helices

Abstract

GWALP23 (acetyl-GGALW5LALALAL12AL14AL16ALW19LAGA-amide) is a useful low-dynamic model peptide for investigations of single-residue influence on protein-lipid interactions and the properties of membrane-spanning helices (J. Biol. Chem. 285, 31723). To investigate the pH dependence, ionization behavior and orientational constraints when potentially negatively charged glutamic acid side chains are present, we have substituted a single Leu residue with Glu at different positions and have incorporated specific 2H-alanines in the core helix or near the ends (Ala-3 and Ala-21) of GWALP23. Solid-state NMR experiments show that the 2H-Ala quadrupolar splittings of core labels change very little over the pH range of 4 to 11.5 when E12, E14 or E16 is present in GWALP23, but show modest shifts above pH 12. The spectra from deuterium labels on alanines 3 and 21 show changes in the unwinding of helix terminals in all three mutants between pH 4.0 and 13. The combined results suggest that these Glu residues may titrate with a pKa near 12 in DLPC and DOPC lipid bilayers. The rather modest shifts in the core alanine 2H quadrupolar splittings, nevertheless, suggest that the orientation of the transmembrane helix actually may change rather little at high pH. Rather, the unwinding of helix ends may be a key indicator of Glu residue titration. Our results are consistent with the expectation that the buried negative charges will aggressively hold their waters of hydration. It is conceivable that the close proximity of E16 to the indole ring of W19 could influence the results.