Influence of Single and Multiple Histidine Residues and their Ionization Properties on Transmembrane Helix Dynamics, Orientations and Fraying

Since aromatic and charged residues are often present in various locations of transmembrane helices of integral membrane proteins, their impacts on the molecular properties of transmembrane proteins and their interactions with lipids are of particular interest in many studies. In this work, I used solid-state deuterium NMR spectroscopy in designed model peptide GWALP23 [GGALW(LA)6LWLAGA] with selective deuterium labels to addresses the pH dependence and influence of single and multiple “guest” histidine residues in the orientation and dynamic behaviors of transmembrane proteins. The mutations include Gly to His (G2/22 to H2/22), Trp to His (W5/19 to H5/19) and Leu to His (L8/16 to H8/16). For the glycine to histidine substitutions, either one or both, the peptides show similar biophysical properties to the host GWALP23 peptide, with modest motional averaging and tilted transmembrane orientations that scale with bilayer thicknesses. Yet, the dynamic motion about the average azimuthal rotation increases significantly when the helix carries only H22. However, when the tryptophan residues, W5 and/or W19 are replaced by histidines, the new histidine residues effectively anchor the transmembrane α-helix, providing similar transmembrane topology. A consistent ~30° shift in helix rotation is observed for Trp to His substitutions and found to be terminal-dependent. Modifying the core sequence of GWALP23 with His residues at positions 8 and 16 provides some interesting insights. The peptide is significantly tilted in DLPC, has multiple orientations in DMPC and surface bound in DPoPC and DOPC lipid bilayers, where the bilayer thicknesses increase consecutively from DLPC to DOPC. Further analysis for peptide with only H8 was performed. Results indicate multiple signal resonances, similar to -H8,16, but in a thicker lipid bilayer. Moreover, the helix with H8 alone significantly responds with pH in DLPC and DMPC lipids and two titration points for H8 was calculated. Finally, mutation of GWALP23 with two adjacent histidines at the N-terminal end (positions 4 and 5) causes a large increase in the motional averaging about helix azimuthal rotation, which in turn obscures the actual orientation and the peptide is found to adopt a very small tilt angle

Comparing Interfacial Trp, Interfacial His and pH Dependence for the Anchoring of Tilted Transmembrane Helical Peptides

Charged and aromatic amino acid residues, being enriched toward the terminals of membrane-spanning helices in membrane proteins, help to stabilize particular transmembrane orientations. Among them, histidine is aromatic and can be positively charge at low pH. To enable investigations of the underlying protein-lipid interactions, we have examined the effects of single or pairs of interfacial histidine residues using the constructive low-dynamic GWALP23 (acetyl-GG2ALW5LALALALALALALW19LAG22A-amide) peptide framework by incorporating individual or paired histidines at locations 2, 5, 19 or 22. Analysis of helix orientation by means of solid-state 2H NMR spectra of labeled alanine residues reveals marked differences with H2,22 compared to W2,22. Nevertheless, the properties of membrane-spanning H2,22WALP23 helices show little pH dependence and are similar to those having Gly, Arg or Lys at positions 2 and 22. The presence of H5 or H19 influences the helix rotational preference but not the tilt magnitude. H5 affects the helical integrity, as residue 7 unwinds from the core helix; yet once again the helix orientation and dynamic properties show little sensitivity to pH. The overall results reveal that the detailed properties of transmembrane helices depend upon the precise locations of interfacial histidine residues