REVERSAL OF THE SURFACE-CHARGE ASYMMETRY IN PURPLE MEMBRANE DUE TO SINGLE AMINO-ACID SUBSTITUTIONS

Twenty-seven mutant bacteriorhodopsin's were screened to determine the pK(a) for reversal of the permanent electric dipole moment. The photo electric response of an aqueous purple-membrane suspension was used to determine the direction of the purple-membrane dipole moment as a fun ction of pH. The pK(a) for the dipole reversal of wild-type bacteriorh odopsin is 4.5. Six of the 27 mutant bacteriorhodopsin's were found to have a pK(a) for dipole reversal larger than that of wild-type bacter iorhodopsin. Two of these mutants, L93T and L93W, involve a neutral am ino acid substitution in the interior of the protein. The direction of the purple-membrane permanent electric dipole moment is determined by the purple-membrane surface charge asymmetry. We conclude that these two substitutions, which do not involve charge replacement, alter the pK(a) for the reversal of the purple-membrane surface charge asymmetry . We suggest that these changes to the pK(a) are due to altered protei n folding at the surface of the purple-membrane induced by single-site substitutions in the protein interior.