X-RAY-DIFFRACTION OF A CYSTEINE-CONTAINING BACTERIORHODOPSIN MUTANT AND ITS MERCURY DERIVATIVE - LOCALIZATION OF AN AMINO-ACID RESIDUE IN THE LOOP OF AN INTEGRAL MEMBRANE-PROTEIN
Mp. Krebs et al., X-RAY-DIFFRACTION OF A CYSTEINE-CONTAINING BACTERIORHODOPSIN MUTANT AND ITS MERCURY DERIVATIVE - LOCALIZATION OF AN AMINO-ACID RESIDUE IN THE LOOP OF AN INTEGRAL MEMBRANE-PROTEIN, Biochemistry, 32(47), 1993, pp. 12830-12834
We have used heavy-atom labeling and X-ray diffraction to localize a s
ingle amino acid in the integral membrane protein bacteriorhodopsin (b
R). To provide a labeling site, we used the bR mutant, A103C, which co
ntains a unique cysteine residue in the short loop between transmembra
ne alpha-helices C and D. The mutant protein was expressed in and puri
fied from Halobacterium halobium, where it forms a two-dimensional cry
stalline lattice. In the lattice form, the protein reacted with the su
lfhydryl-specific reagent p-chloromercuribenzoate (p-CMB) in a 1:0.9 s
toichiometry to yield the p-mercuribenzoate derivative (A103C-MB). The
functional properties of A103C and A103C-MB, including the visible ab
sorption spectrum, light-dark adaptation, photocycle, and proton relea
se kinetics, were similar to those of wild-type bR. X-ray diffraction
experiments demonstrated that A103C and A103C-MB membranes have the sa
me hexagonal protein lattice as wild-type purple membrane. Thus, neith
er the cysteine substitution nor mercury labeling detectably affected
bR structure or function. By using Fourier difference methods, the in-
plane position of the mercuribenzoate label was calculated from intens
ity differences in the X-ray diffraction patterns of A103C and A103C-M
B. This analysis revealed a well-defined mercury peak located between
alpha-helices C and D. The approach reported here offers promise for r
efining the bR structural model, for monitoring conformational changes
in bR photointermediates, and for studying the structure of other pro
teins in two-dimensional crystals.