title: Ellman's reagent in promoting crystallization and structure determination of Anabaena CcbP.
abstract: Obtaining crystals presented a bottleneck in the structural study of Anabaena cyanobacterial Ca2+-binding protein (CcbP). In this report, the promoting effect of Ellman's reagent [5,5'-dithiobis(2-nitrobenzoic acid); DTNB] on the crystallization of CcbP is described. CcbP contains one free cysteine. A quick and simple oxidation reaction with DTNB blocked the free cysteine in purified CcbP and generated a homogenous monomeric protein for crystallization. The crystal structure of DTNB-modified CcbP was determined by the single-wavelength anomalous diffraction method. Structure analysis indicated that DTNB modification facilitated crystallization of CcbP by inducing polar interactions in the crystal lattice. DTNB-mediated cysteine modification was demonstrated to have little effect on the overall structure and the Ca2+ binding of CcbP. Thus, DTNB modification may provide a simple and general approach for protein modification to improve the success of crystallization screening.
authors.affiliation: State Key Laboratory of Protein and Plant Gene Research, and Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, No. 5 Yiheyuan Road, Beijing 100871, People's Republic of China.
authors.forename: Xue-Xin
authors.initials: XX
authors.lastname: Fan
authors.forename: Yan-Feng
authors.initials: YF
authors.lastname: Zhou
authors.forename: Xiang
authors.initials: X
authors.lastname: Liu
authors.forename: Lan-Fen
authors.initials: LF
authors.lastname: Li
authors.forename: Xiao-Dong
authors.initials: XD
authors.lastname: Su
mesh_terms: D017033:Anabaena; D001426:Bacterial Proteins / Q000737:chemistry*; D002118:Calcium / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D003460:Crystallization / Q000379:methods; D018360:Crystallography, X-Ray; D004228:Dithionitrobenzoic Acid / Q000737:chemistry*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017186:Titrimetry
chemical_list: D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins; D004228:Dithionitrobenzoic Acid; D002118:Calcium