Membrane Physical Properties Influence Transmembrane Helix Formation

Abstract

ThepHLIPpeptidehasthreestates:(I)solubleinaqueousbuffer,(II)bound to the bilayer surface at neutral pH, and (III) inserted as atransmembrane (TM) helix at acidic pH. The membrane insertion ofpHLIP at low pH can be used to target the acidic tissues character-istic of different diseases, such as cancer. We find that the α-helixcontent of state II depends on lipid acyl chain length but notcholesterol, suggesting the helicity of the bound state may be con-trolled by the bilayer elastic bending modulus. Experiments withthe P20G variant show the proline residue in pHLIP reduces theα-helix content of both states II and III. We also observe that themembrane insertion pKa is influencedbymembranephysicalprop-erties, following a biphasic pattern similar to the membrane thick-ness optima observed for the function of eukaryotic membraneproteins. Because tumor cells exhibit altered membrane fluidity,we suggest this might influence pHLIP tumor targeting. We useda cell insertion assay to determine the pKa in live cells, observingthat the properties in liposomes held in the more complex plasmamembrane. Our results show that the formation of a TM helix ismodulated by both the conformational propensities of the peptideand the physical properties of the bilayer. These results suggest aphysical role for helix-membrane interactions in optimizing thefunction of more complex TM proteins.