title: Crystallization and preliminary X-ray crystallographic analysis of a blue-light-absorbing proteorhodopsin.
abstract: Proteorhodopsins (PRs), seven-transmembrane chromoproteins with retinal as a chromophore, are light-driven proton pumps. To elucidate the light-driven proton-pumping mechanism of PRs, a pET28a vector containing the blue-light-absorbing proteorhodopsin (BPR) gene was constructed and the protein was overexpressed in Escherichia coli. The protein was purified by immobilized metal-ion affinity chromatography (IMAC). The purified BPR D97N mutant protein (BPR_D97N) was crystallized using the vapour-diffusion method. Preliminary X-ray diffraction data analysis showed that the crystal belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 161.6, b = 168.6, c = 64.7 Å. A complete data set was collected to 3.3 Å resolution using synchrotron radiation on beamline X06 of the Swiss Light Source (SLS). Molecular replacement was unsuccessful. To solve the structure of BPR_D97N by experimental phasing, selenomethionine-substituted protein crystals were prepared. These crystals diffracted to 3.0 Å resolution and a complete data set was collected on beamline BL17U of the Shanghai Synchrotron Radiation Facility (SSRF). Heavy-atom substructure determination and phasing by SAD clearly showed that the crystal contained five molecules in the asymmetric unit, with a V(M) of 3.26 Å(3) Da(-1) and a solvent content of 62.3%.
authors.affiliation: Key Laboratory of Agricultural and Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, People's Republic of China.
authors.forename: Ning
authors.initials: N
authors.lastname: Wang
authors.forename: Meitian
authors.initials: M
authors.lastname: Wang
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Gao
authors.forename: Tingting
authors.initials: T
authors.lastname: Ran
authors.forename: Yanli
authors.initials: Y
authors.lastname: Lan
authors.forename: Jian
authors.initials: J
authors.lastname: Wang
authors.forename: Langlai
authors.initials: L
authors.lastname: Xu
authors.forename: Weiwu
authors.initials: W
authors.lastname: Wang
mesh_terms: D003460:Crystallization; D018360:Crystallography, X-Ray; D009154:Mutation; D012243:Rhodopsin / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D025604:Rhodopsins, Microbial
chemical_list: D025604:Rhodopsins, Microbial; C413548:proteorhodopsin; D012243:Rhodopsin