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

   https://doi.org/10.3390/biom10020273  

   Afrose, Fahmida; Koeppe II, Roger E. (February 2020, Biomolecules)

   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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2. Bioinspired Design Rules for Flipping across the Lipid Bilayer from Systematic Simulations of Membrane Protein Segments

   https://doi.org/10.1039/D5ME00032G  

   Park, ByungUk; Van_Lehn, Reid C (May 2025, Molecular Systems Design & Engineering)

   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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3. Tight hydrophobic core and flexible helices yield MscL with a high tension gating threshold and a membrane area mechanical strain buffer

   https://doi.org/10.3389/fchem.2023.1159032  

   Sharma, Arjun; Anishkin, Andriy; Sukharev, Sergei; Vanegas, Juan M. (May 2023, Frontiers in Chemistry)

   The mechanosensitive (MS) channel of large conductance, MscL, is the high-tension threshold osmolyte release valve that limits turgor pressure in bacterial cells in the event of drastic hypoosmotic shock. Despite MscL from Mycobacterium tuberculosis (TbMscL) being the first structurally characterized MS channel, its protective mechanism of activation at nearly-lytic tensions has not been fully understood. Here, we describe atomistic simulations of expansion and opening of wild-type (WT) TbMscL in comparison with five of its gain-of-function (GOF) mutants. We show that under far-field membrane tension applied to the edge of the periodic simulation cell, WT TbMscL expands into a funnel-like structure with trans-membrane helices bent by nearly 70°, but does not break its ‘hydrophobic seal’ within extended 20 μs simulations. GOF mutants carrying hydrophilic substitutions in the hydrophobic gate of increasing severity (A20N, V21A, V21N, V21T and V21D) also quickly transition into funnel-shaped conformations but subsequently fully open within 1–8 μs. This shows that solvation of the de-wetted (vapor-locked) constriction is the rate-limiting step in the gating of TbMscL preceded by area-buffering silent expansion. Pre-solvated gates in these GOF mutants reduce this transition barrier according to hydrophilicity and the most severe V21D eliminates it. We predict that the asymmetric shape-change of the periplasmic side of the channel during the silent expansion provides strain-buffering to the outer leaflet thus re-distributing the tension to the inner leaflet, where the gate resides. 

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4. Biophysics of pH-Driven Membrane Insertion: A Review of the pHLIP Peptide

   https://doi.org/10.1021/acs.jpcb.5c00225  

   Maia, Raiza; Ataka, Kenichi; Heberle, Joachim; Baiz, Carlos R (May 2025, The Journal of Physical Chemistry B)

   The pH Low Insertion Peptide (pHLIP) is a useful model for exploring the biophysical chemistry of pH-driven membrane insertion and folding. This review discusses recent advancements in understanding the molecular mechanisms underlying pHLIP behavior, focusing on its ability to transition from a soluble, unstructured state to a membrane-inserted α-helix. Protonation of acidic residues, changes in peptide hydrophobicity, and interactions with the lipid bilayer, are described. Recent studies using NMR, infrared spectroscopy, and molecular dynamics simulations have provided a stepwise mechanistic model of the coupled folding and insertion process including its intermediate states present under different pH conditions. In addition, pHLIP ability to selectively target acidic microenvironments, such as those found in tumors, has made it a promising tool for biomedical applications. We provide an overview of recent fundamental studies and applications and discuss how future work can benefit from combining advanced experimental and computational approaches to refine our understanding of the peptide’s structure–function relationships. 

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5. An Amphipathic Alpha-Helix Domain from Poliovirus 2C Protein Tubulate Lipid Vesicles

   https://doi.org/10.3390/v12121466  

   Varkey, Jobin; Zhang, Jiantao; Kim, Junghyun; George, Gincy; He, Guijuan; Belov, George; Langen, Ralf; Wang, Xiaofeng (December 2020, Viruses)

   null (Ed.)

   Positive-strand RNA viruses universally remodel host intracellular membranes to form membrane-bound viral replication complexes, where viral offspring RNAs are synthesized. In the majority of cases, viral replication proteins are targeted to and play critical roles in the modulation of the designated organelle membranes. Many viral replication proteins do not have transmembrane domains, but contain single or multiple amphipathic alpha-helices. It has been conventionally recognized that these helices serve as an anchor for viral replication protein to be associated with membranes. We report here that a peptide representing the amphipathic α-helix at the N-terminus of the poliovirus 2C protein not only binds to liposomes, but also remodels spherical liposomes into tubules. The membrane remodeling ability of this amphipathic alpha-helix is similar to that recognized in other amphipathic alpha-helices from cellular proteins involved in membrane remodeling, such as BAR domain proteins. Mutations affecting the hydrophobic face of the amphipathic alpha-helix severely compromised membrane remodeling of vesicles with physiologically relevant phospholipid composition. These mutations also affected the ability of poliovirus to form plaques indicative of reduced viral replication, further underscoring the importance of membrane remodeling by the amphipathic alpha-helix in possible relation to the formation of viral replication complexes. 

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