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 single amino acid in the integral membrane protein bacteriorhodopsin (bR). To provide a labeling site, we used the bR mutant, A103C, which contains a unique cysteine residue in the short loop between transmembrane alpha-helices C and D. The mutant protein was expressed in and purified from Halobacterium halobium, where it forms a two-dimensional crystalline lattice. In the lattice form, the protein reacted with the sulfhydryl-specific reagent p-chloromercuribenzoate (p-CMB) in a 1:0.9 stoichiometry to yield the p-mercuribenzoate derivative (A103C-MB). The functional properties of A103C and A103C-MB, including the visible absorption spectrum, light-dark adaptation, photocycle, and proton release kinetics, were similar to those of wild-type bR. X-ray diffraction experiments demonstrated that A103C and A103C-MB membranes have the same hexagonal protein lattice as wild-type purple membrane. Thus, neither 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 intensity differences in the X-ray diffraction patterns of A103C and A103C-MB. This analysis revealed a well-defined mercury peak located between alpha-helices C and D. The approach reported here offers promise for refining the bR structural model, for monitoring conformational changes in bR photointermediates, and for studying the structure of other proteins in two-dimensional crystals.
Krebs
M P
MP
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Behrens
W
W
Mollaaghababa
R
R
Khorana
H G
HG
Heyn
M P
MP
D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000031:analogs & derivatives / Q000737:chemistry* / Q000235:genetics; D002729:Chloromercuribenzoates / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D005583:Fourier Analysis; D019617:Halobacterium salinarum / Q000737:chemistry* / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D014961:X-Ray Diffraction
D002729:Chloromercuribenzoates; D008565:Membrane Proteins; D011994:Recombinant Proteins; D001436:Bacteriorhodopsins; D003545:Cysteine