PERTURBED INTERACTION BETWEEN RESIDUE-85 AND RESIDUE-204 IN TYR-185-]PHE AND ASP-85-]GLU BACTERIORHODOPSINS

According to earlier reports, residue 85 in the bacteriorhodopsin muta nts D85E and Y185F deprotonates with two apparent pK(a) values. Additi onally, in Y185F, Asp-85 becomes significantly more protonated during light adaptation. We provide a new explanation for these findings. It is based on the scheme that links the protonation state of residue 85 to the protonation state of residue 204 (S.P. Balashov, E.S. Imasheva, R. Govindjee, and T.G. Ebrey. 1996. Biophys. J. 70:473-481; H.T. Rich ter, L.S. Brown, R. Needleman, and J.K. Lanyi. 1996. Biochemistry. 35: 4054-4062) and justified by the observation that the biphasic titratio n curves of D85E and Y185F are converted to monophasic when the E204Q residue change is introduced as a second mutation. Accordingly, the D8 5E and Y185F mutations are not the cause of the biphasic titration, as that is a property of the wild-type protein. By perturbing the extrac ellular region of the protein, the mutations increase the pK(a) of res idue 85. This increases the amplitude of the second titration componen t and makes the biphasic character of the curves more obvious. Likewis e, a small rise in the pK(a) of Asp-85 when the retinal isomerizes fro m 13-cis,15-syn to all-trans accounts for the changed titration behavi or of Y185F after light adaptation. This mechanism simplifies and unit es the interpretation of what had appeared to be complex and unrelated phenomena.