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

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.