Investigating Possible Interactions between Ionizable Residues in Model Transmembrane Peptides

Abstract

The study of charged residues in the hydrophobic interior of transmembrane proteins is crucial to the understanding of membrane proteins and their function. To this end, we have employed GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide), a constructive low-dynamic model peptide, for investigations of single-residue influence on protein-lipid interactions (JBC 285, 31723). We have substituted a single Leu residue at position 12, within the hydrophobic core of the GWALP23 sequence, with either Arg (R12) or Glu (E12). Specific 2H-labeled Ala residues have been incorporated within the -R12 sequence for detection by solid-state 2H NMR. GWALP23-R12 remains charged in DOPC bilayers, even under strongly alkaline pH conditions, and displays multi-state behavior (JACS 132, 5803). To determine if the presence of an adjacent peptide with Glu at position 12 would influence the behavior of the -R12 peptide, we incorporate a mixture of labeled GWALP23-R12 and unlabeled -E12 peptides in DOPC bilayers. Preliminary results for oriented samples with both peptides suggest that -R12 remains multi-state in the presence of the -E12 peptide in DOPC bilayers at pH 6. By contrast, oriented samples of -R12 in DLPC bilayers show clearly defined quadrupolar splittings. In the presence of -E12, we observe small changes in the quadrupolar splittings, indicative of changes in orientation of the -R12 peptide. It is possible that peptide crowding within the bilayer may influence ionic interactions between the -E12 and -R12 peptides; therefore we are currently investigating the effect of varying the peptide/peptide and peptide/lipid ratios. Similar experiments with GW3,21ALP23-R12, with the Trp residues moved to the opposite face of the helix, also suggest interaction between -E12 and -R12 in both DLPC and DOPC bilayers at pH 6. Additionally, we are studying possible ion-pair interactions in a peptide with two ionizable residues, GWALP23-R12,E16.