Perturbed interaction between residues 85 and 204 in Tyr-185-->Phe and Asp-85-->Glu bacteriorhodopsins.
According to earlier reports, residue 85 in the bacteriorhodopsin mutants D85E and Y185F deprotonates with two apparent pKa values. Additionally, 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. Richter, L.S. Brown, R. Needleman, and J.K. Lanyi. 1996. Biochemistry. 35:4054-4062) and justified by the observation that the biphasic titration curves of D85E and Y185F are converted to monophasic when the E204Q residue change is introduced as a second mutation. Accordingly, the D85E and Y 185F mutations are not the cause of the biphasic titration, as that is a property of the wild-type protein. By perturbing the extracellular region of the protein, the mutations increase the pKa of residue 85. This increases the amplitude of the second titration component and makes the biphasic character of the curves more obvious. Likewise, a small rise in the pKa of Asp-85 when the retinal isomerizes from 13-cis, 15-syn to all-trans accounts for the changed titration behavior of Y185F after light adaptation. This mechanism simplifies and unites the interpretation of what had appeared to be complex and unrelated phenomena.
Richter
H T
HT
Department of Physiology and Biophysics, University of California, Irvine 92717, USA.
Needleman
R
R
Lanyi
J K
JK
D001224:Aspartic Acid; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism*; D003624:Darkness; D018698:Glutamic Acid; D006217:Halobacterium / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D014443:Tyrosine
D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D014443:Tyrosine; D010649:Phenylalanine; D001436:Bacteriorhodopsins