title: Wild-type and mutant bacteriorhodopsins D85N, D96N, and R82Q: purification to homogeneity, pH dependence of pumping, and electron diffraction.
abstract: Bacterioopsin, expressed in Escherichia coli as a fusion protein with 13 heterologous residues at the amino terminus, has been purified in the presence of detergents and retinylated to give bacteriorhodopsin. Further purification yielded pure bacteriorhodopsin, which had an absorbance ratio (A280/A lambda max) of 1.5 in the dark-adapted state in a single-detergent environment. This protein has a folding rate, absorbance spectrum, and light-induced proton pumping activity identical with those of bacteriorhodopsin purified from Halobacterium halobium. Protein expressed from the mutants D85N, D96N, and R82Q and purified similarly yielded pure protein with absorbance ratios of 1.5. Proton pumping rates of bacteriorhodopsins with the wild-type sequence and variants D85N, D96N, and R82Q were determined in phospholipid vesicles as a function of pH. D85N was inactive at all pH values, whereas D96N was inactive from pH 7.0 to pH 8.0, where wild type is most active, but had some activity at low pH. R82Q showed diminished proton pumping with the same pH dependence as for wild type. Bacteriorhodopsin purified from E. coli crystallized in two types of two-dimensional crystal lattices suitable for low-dose electron diffraction, which permit detailed analysis of structural differences in site-directed variants. One lattice was trigonal, as in purple membrane, and showed a high-resolution electron diffraction pattern from glucose-sustained patches. The other lattice was previously uncharacterized with unit cell dimensions a = 127 A, b = 67 A, and symmetry of the orthorhombic plane group pgg.
authors.affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
authors.forename: L J
authors.initials: LJ
authors.lastname: Miercke
authors.forename: M C
authors.initials: MC
authors.lastname: Betlach
authors.forename: A K
authors.initials: AK
authors.lastname: Mitra
authors.forename: R F
authors.initials: RF
authors.lastname: Shand
authors.forename: S K
authors.initials: SK
authors.lastname: Fong
authors.forename: R M
authors.initials: RM
authors.lastname: Stroud
mesh_terms: D001436:Bacteriorhodopsins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000648:ultrastructure; D003460:Crystallization; D004583:Electrons; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D008854:Microscopy, Electron; D009154:Mutation; D011522:Protons; D013056:Spectrophotometry, Ultraviolet
chemical_list: D011522:Protons; D001436:Bacteriorhodopsins