Crystallization and preliminary X-ray crystallographic analysis of a blue-light-absorbing proteorhodopsin

• Published in: Acta crystallographica. Section F, Structural biology and crystallization communications Vol. 68; no. 3; pp. 281 - 283
• Main Authors: Wang, Ning, Wang, Meitian, Gao, Yanyan, Ran, Tingting, Lan, Yanli, Wang, Jian, Xu, Langlai, Wang, Weiwu
• Format: Journal Article
• Language: English
• Published: 5 Abbey Square, Chester, Cheshire CH1 2HU, England International Union of Crystallography 01.03.2012
• Subjects: BPR; Crystallization; Crystallization Communications; Crystallography; Crystallography, X-Ray; Crystals; Diffraction; Mutation; Phasing; Proteins; proteorhodopsins; Rhodopsin - analysis; Rhodopsin - chemistry; Rhodopsin - genetics; Rhodopsins, Microbial; Synchrotron radiation; X-rays
• ISSN: 1744-3091; 1744-3091
• DOI: 10.1107/S1744309111043612

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 P21212, 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 VM of 3.26 Å3 Da−1 and a solvent content of 62.3%.