Probing of the retinal binding site of bacteriorhodopsin by affinity labeling.
The position of the chromophore within bacteriorhodopsin has been identified by cross-linking a cysteine group, introduced by site-specific mutagenesis, with a chromophore suitably derivatized with an active leaving group. Since bacteriorhodopsin has no cysteines, a site-specific cysteine mutant will contain only one free sulfhydryl group capable of reacting with the retinal analog. Met118, Thr121, and Ser141 were selected to be mutated to cysteine. No pigment absorbing in the visible region was obtained for the Ser141Cys mutant. The Met118Cys and Thr121Cys mutants have similar absorption maxima, proton pumping efficiencies and photocycles to those of the wild-type pigment. 4-Bromoretinal, in which the reactive allylic halide readily undergoes nucleophilic displacement, was used as the reactive chromophore. Pigments were obtained on reaction of all-trans-4-bromoretinal with the apoproteins of Met118Cys, Thr121Cys, and wild-type bacteriorhodopsin (lambda max = 464-470 nm). Analysis of the denatured pigments on SDS-polyacrylamide gels showed incorporation of tritiated chromophore into the Met118Cys mutant but not into the wild-type or Th4121Cys pigments. Met118Cys apoprotein which was preincubated with the cysteine-specific reagent N-ethylmaleimide formed a pigment with 4-bromoretinal but no cross-linking was observed, providing evidence that the cross-linking of the chromophore is to the cysteine at 118. We conclude that Met118 is positioned in the chromophore binding pocket, proximal to the C-4 position of cyclohexyl ring of retinal.
Feng
Y
Y
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston 29425.
Menick
D R
DR
Katz
B M
BM
Beischel
C J
CJ
Hazard
E S
ES
Misra
S
S
Ebrey
T G
TG
Crouch
R K
RK
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D019617:Halobacterium salinarum / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017494:Proton Pumps; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012172:Retinaldehyde / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D017422:Sequence Analysis, DNA
C089628:4-bromoretinal; D000345:Affinity Labels; D003432:Cross-Linking Reagents; D017494:Proton Pumps; D011994:Recombinant Proteins; D001436:Bacteriorhodopsins; D003545:Cysteine; D012172:Retinaldehyde