The retinylidene Schiff base counterion in bacteriorhodopsin.
Previous studies of bacteriorhodopsin have indicated interactions between Asp-85, Asp-212, Arg-82, and the retinylidene Schiff base. The counterion environment of the Schiff base has now been further investigated by using single and double mutants of the above amino acids. Chromophore regeneration from bacterioopsin proceeds to a normal extent in the presence of a single aspartate or glutamate residue at position 85 or 212, whereas replacement of both charged amino acids in the mutant Asp-85----Asn/Asp-212----Asn abolishes the binding of retinal. This indicates that a carboxylate group at either residue 85 or 212 is required as counterion for formation and for stabilization of the protonated Schiff base. Measurements of the pKa of the Schiff base reveal reductions of greater than 3.5 units for neutral single mutants of Asp-85 but only decreases of less than 1.2 units for corresponding substitutions of Asp-212, relative to the wild type. Substitutions of Asp-85 show large red shifts in the absorption spectrum that are partially reversible upon addition of anions, whereas mutants of Asp-212 display minor red shifts or blue shifts. We conclude, therefore, that Asp-85 is the retinylidene Schiff base counterion in wild-type bacteriorhodopsin. In the mutant Asp-85----Asn/Asp-212----Asn formation of a protonated Schiff base chromophore is restored in the presence of salts. The spectral properties of the double mutant are similar to those of the acid-purple form of bacteriorhodopsin. Upon addition of salts the folded structure of wild-type and mutant proteins can be stabilized at low pH in lipid/detergent micelles. The data indicate that exogenous anions serve as surrogate counterions to the protonated Schiff base, when the intrinsic counterions have been neutralized by mutation or by protonation.
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
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T
Marti
S J
SJ
Rösselet
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H
Otto
M P
MP
Heyn
H G
HG
Khorana
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics; D004563:Electrochemistry; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D009154:Mutation; D012176:Retinoids / Q000737:chemistry*; D012545:Schiff Bases / Q000737:chemistry; D012965:Sodium Chloride; D013057:Spectrum Analysis
D007477:Ions; D012176:Retinoids; D012545:Schiff Bases; D012965:Sodium Chloride; D001436:Bacteriorhodopsins