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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.
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This content will become publicly available on May 9, 2026
Bioinspired Design Rules for Flipping across the Lipid Bilayer from Systematic Simulations of Membrane Protein Segments
The orientation of integral membrane proteins (IMPs) with respect to the membrane is established during protein synthesis and insertion into the membrane. After synthesis, IMP orientation is thought to be fixed due to the thermodynamic barrier for “flipping” protein loops or helices across the hydrophobic core of the membrane in a process analogous to lipid flip-flop. A notable exception is EmrE, a homodimeric IMP with an N-terminal transmembrane helix that can flip across the membrane until flipping is arrested upon dimerization. Understanding the features of the EmrE sequence that permit this unusual flipping behavior would be valuable for guiding the design of synthetic materials capable of translocating or flipping charged groups across lipid membranes. To elucidate the molecular mechanisms underlying flipping in EmrE and derive bioinspired design rules, we employ atomistic molecular dynamics simulations and enhanced sampling techniques to systematically investigate the flipping of truncated segments of EmrE. Our results demonstrate that a membrane-exposed charged glutamate residue at the center of the N-terminal helix lowers the energetic barrier for flipping (from ~12.1 kcal mol-1 to ~5.4 kcal mol-1) by stabilizing water defects and minimizing membrane perturbation. Comparative analysis reveals that the marginal hydrophobicity of this helix, rather than the marginal hydrophilicity of its loop, is the key determinant of flipping propensity. Our results further indicate that interhelical hydrogen bonding upon dimerization inhibits flipping. These findings establish several bioinspired design principles to govern flipping in related materials: (1) marginally hydrophobic helices with membrane-exposed charged groups promote flipping, (2) modulating protonation states of membrane-exposed groups tunes flipping efficiency, and (3) interhelical hydrogen bonding can be leveraged to arrest flipping. These insights provide a foundation for engineering synthetic peptides, engineered proteins, and biomimetic nanomaterials with controlled flipping or translocation behavior for applications in intracellular drug delivery and membrane protein design.
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- Award ID(s):
- 1817292
- PAR ID:
- 10589044
- Publisher / Repository:
- Royal Society of Chemistry
- Date Published:
- Journal Name:
- Molecular Systems Design & Engineering
- ISSN:
- 2058-9689
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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