Open AccessEllman's reagent in promoting crystallization and structure determination of Anabaena CcbP
Author(s) -
Fan XueXin,
Zhou YanFeng,
Liu Xiang,
Li LanFen,
Su XiaoDong
Publication year - 2012
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309112034938
Subject(s) - dtnb , crystallization , reagent , cysteine , chemistry , protein crystallization , crystallography , anabaena , biochemistry , organic chemistry , enzyme , glutathione , biology , cyanobacteria , bacteria , genetics
Obtaining crystals presented a bottleneck in the structural study of Anabaena cyanobacterial Ca 2+ ‐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 Ca 2+ binding of CcbP. Thus, DTNB modification may provide a simple and general approach for protein modification to improve the success of crystallization screening.