Insertion of Bacteriorhodopsin Helix C Variants into Biological Membranes

[Image: see text] A peptide corresponding to bacteriorhodopsin (bR) helix C, later named pHLIP, inserts across lipid bilayers as a monomeric α-helix at acidic pH, but is an unstructured surface-bound monomer at neutral pH. As a result of such pH-responsiveness, pHLIP targets acidic tumors and has been used as a vehicle for imaging and drug-delivery cargoes. To gain insights about the insertion of bR helix C into biological membranes, we replaced two key aspartic residues that control the topological transition from the aqueous phase into a lipid bilayer. Here, we used an in vitro transcription–translation system to study the translocon-mediated insertion of helix C-derived segments into rough microsomes. Our data provide the first quantitative biological understanding of this effect. Interestingly, replacing the aspartic residues by glutamic residues does not significantly alters the insertion propensity, while replacement by alanines promotes a transmembrane orientation. These results are consistent with mutational data obtained in synthetic liposomes by manipulating pH conditions. Our findings support the notion that the translocon facilitates topogenesis under physiological pH conditions.