Membrane physical properties influence transmembrane helix formation

Abstract

The pHLIP peptide has three states: (I) soluble in aqueous buffer, (II) bound to the bilayer surface at neutral pH, and (III) inserted as a transmembrane (TM) helix at acidic pH. The membrane insertion of pHLIP at low pH can be used to target the acidic tissues characteristic of different diseases, such as cancer. We find that the α-helix content of state II depends on lipid acyl chain length but not cholesterol, suggesting the helicity of the bound state may be controlled by the bilayer elastic bending modulus. Experiments with the P20G variant show the proline residue in pHLIP reduces the α-helix content of both states II and III. We also observe that the membrane insertion pKa is influenced by membrane physical properties, following a biphasic pattern similar to the membrane thickness optima observed for the function of eukaryotic membrane proteins. Because tumor cells exhibit altered membrane fluidity, we suggest this might influence pHLIP tumor targeting. We used a cell insertion assay to determine the pKa in live cells, observing that the properties in liposomes held in the more complex plasma membrane. Our results show that the formation of a TM helix is modulated by both the conformational propensities of the peptide and the physical properties of the bilayer. These results suggest a physical role for helix-membrane interactions in optimizing the function of more complex TM proteins.alpha helix | peptide folding | tumor acidity | bilayer thickness | vesicle properties

Aspartate Embedding Depth Affects pHLIP’s Insertion pKa

“…This concept of using simple hydrophobicity calculations of the putative spanning domain to correctly predict the change in direction of the insertion pKa is something we have also done in other recent work 19 using membranes of different acyl chain length. In this other work, we found that MPEx results were also good predictors of pHLIP’s insertion pKa in lipids of short to intermediate core thicknesses, leaving some hope that the semblance of an average may yet have practical use in our system.…”

Targeting acidity in diseased tissues: Mechanism and applications of the membrane-inserting peptide, pHLIP

“…The proline-20 to glycine variant exhibited α-helical structure in both State II and State III, leading to promiscuous insertion activity at a broad pH range, with a far lower apparent cooperativity of insertion than WT pHLIP (35). Proline, known commonly to destabilize helices due to its conformational constraints, is likely to limit the α-helicity of pHLIP peptides in States I and II, maintaining a high energy barrier to membrane insertion until protonation of the transmembrane acidic residues promotes helix formation during the State II to State III transition.…”

“…While the longer acyl-chain liposomes exhibit an increased stiffness, cholesterol concentration has no effect on the bending modulus of monounsaturated membranes (71, 72). A likely conclusion, therefore, is that the elastic properties of the bilayer influence helix formation on the membrane surface (35). Cell experiments suggest that membrane fluidity is more determinant that membrane thickness in controlling pHLIP’s pK ins .…”

Mapping Allostery through Computational Glycine Scanning and Correlation Analysis of Residue–Residue Contacts

“…3,4 Thus, these macromolecules are able to switch their conformations upon sensing environmental perturbations. There are a variety of such perturbations, such as interaction with another macromolecule, 1,5 interaction with a ligand, 6 solvent conditions 7–10 -(polarity and pH), physical environment 11–15 (salt, pressure, temperature, and light), or post-translational modifications. 13,16 …”