Rational design of 'water-soluble' bacteriorhodopsin variants.
We have explored the interchangeability of soluble and membrane proteins by attempting to render a helical membrane protein 'water soluble' through mutation of its lipid-exposed residues. Using an atomic resolution structure of bacteriorhodopsin (bR), two different strategies were developed to identify lipid-exposed residues for mutation. In the first strategy all residues in trimeric bR with solvent accessibility >35% were marked for replacement. Replacement residues were chosen so as to map an average surface of helical soluble proteins onto the bR surface, resulting in the mutagenesis of 14.9% of surface residues. The second strategy took into account the observation that accessible residues can be categorized as fully or partially accessible. Consequently, three mutants were designed based on monomeric bR, all with their accessible residues changed and with varying extents of mutagenesis of partially accessible residues. 13.5-24.3% of the wild-type surface was altered in these designs. The construct for the first design was cloned into Escherichia coli. Trace amounts of the mutant protein were expressed with the concurrent overexpression of an endogenous prolyl isomerase. In contrast, all three mutant proteins of the second design expressed well and could be purified to homogeneity. Systematic refolding trials were undertaken with limited success at solubilization in aqueous media. We have discussed the feasibility of applying the 'solubilization strategy' outlined here to membrane proteins.
Mitra
Kakoli
K
Department of Molecular Biophysics and Biochemistry, the Howard Hughes Medical Institute, Yale University, 266 Whitney Avenue, New Haven, CT 06520-8114, USA.
Steitz
Thomas A
TA
Engelman
Donald M
DM
D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D016415:Sequence Alignment; D012995:Solubility; D014867:Water
D014867:Water; D001436:Bacteriorhodopsins