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 o f transmembrane proteins using site-directed mutagenesis represents a novel approach to render membrane proteins more soluble in aqueous sol ution. We have sequentially introduced as many as five polar and charg ed amino acids onto the lipid-exposed face of helix D of bacteriorhodo psin from Halobacterium salinarium. The most polar mutant (Q4D) has fo ur glutamine residues at positions 113, 116, 120 and 124 and an aspart ate at position 117. In combination with wild-type residues Gln105, Th r107, Thr121 and Thr128, the Q4D mutant has a nearly uninterrupted str ipe of polar residues on the surface of helix D, All of the mutants re fold, bind retinal and the resulting pigments exhibit light-and dark-a dapted 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 d emonstrate that micelle-solubilized bacteriorhodopsin can tolerate mul tiple non-conservative substitution of amino acids that face the non-p olar portion of the lipid bilayer in vivo, thus lending credence to th e notion of partial or complete solubilization of integral membrane pr oteins by site-directed mutagenesis.