Reduction of membrane protein hydrophobicity by site-directed mutagenesis: introduction of multiple polar residues in helix D of bacteriorhodopsin.

Introduction of polar and charged residues on the lipid-exposed face of transmembrane proteins using site-directed mutagenesis represents a novel approach to render membrane proteins more soluble in aqueous solution. We have sequentially introduced as many as five polar and charged amino acids onto the lipid-exposed face of helix D of bacteriorhodopsin from Halobacterium salinarium. The most polar mutant (Q4D) has four glutamine residues at positions 113, 116, 120 and 124 and an aspartate at position 117. In combination with wild-type residues Gln105, Thr107, Thr121 and Thr128, the Q4D mutant has a nearly uninterrupted stripe of polar residues on the surface of helix D. All of the mutants refold, bind retinal and the resulting pigments exhibit light- and dark-adapted UV and visible spectroscopic properties that are similar to the wild-type pigment, indicating that the secondary, tertiary and active site structures are similar to the wild-type protein. These results demonstrate that micelle-solubilized bacteriorhodopsin can tolerate multiple non-conservative substitution of amino acids that face the non-polar portion of the lipid bilayer in vivo, thus lending credence to the notion of partial or complete solubilization of integral membrane proteins by site-directed mutagenesis.