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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. 

To address biophysical principles and lipid interactions that underlie the properties of membrane proteins, modifications that vary the neighbors of tryptophan residues in the highly dynamic transmembrane helix of GW^4,20ALP23 (acetyl‐GGAW⁴A(LA)₆LAW²⁰AGA‐amide) were examined using deuterium NMR spectroscopy. It was found that L^5,19GW^4,20ALP23, a sequence isomer of the low to moderately dynamic GW^5,19ALP23, remains highly dynamic. By contrast, a removal of W4 to produce F^4,5GW²⁰ALP23 restores a low level of dynamic averaging, similar to that of the F^4,5GW¹⁹ALP23 helix. Interestingly, a high level of dynamic averaging requires the presence of both tryptophan residues W4 and W20, on opposite faces of the helix, and does not depend on whether residue 5 is Leu or Ala. Aspects of helix unwinding and potential oligomerization are discussed with respect to helix dynamic averaging and the locations of particular residues at a phosphocholine membrane interface.