PicW2 from Picea wilsonii: preparation, purification, crystallization and X-ray diffraction analysis

Low temperature is a major limiting factor for plant growth and development. Dehydrin proteins are generally induced in response to low-temperature stress. In previous research, a full-length dehydrin gene, PicW2, was isolated from Picea wilsonii and its expression was associated with hardiness to cold. In order to gain insight into the mechanism of low-temperature tolerance by studying its three-dimensional crystal structure, prokaryotically expressed PicW2 dehydrin protein was purified using chitosan-affinity chromatography and gel filtration, and crystallized using the vapour-diffusion method. The crystal grew in a condition consisting of 0.1   M HEPES pH 8.0, 25%(w/v) PEG 3350 using 4   mg   ml − 1 protein solution at 289   K. X-ray diffraction data were collected from a crystal at 100   K to 2.82   Å resolution. The crystal belonged to space group C121, with unit-cell parameters a = 121.55, b = 33.26, c = 73.39   Å , α = γ = 90.00, β   = 109.01 ° . The asymmetric unit contained one molecule of the protein, with a corresponding Matthews coefficient of 2.87   Å 3   Da − 1 and a solvent content of 57.20%. Owing to a lack of structures of homologous dehydrin proteins, molecular-replacement trials failed. Data collection for selenium derivatization of PicW2 and crystal structure determination is currently in progress.
Source: Acta Crystallographica Section F - Category: Biochemistry Authors: Tags: PicW2 dehydrin Picea wilsonii crystallization low-temperature tolerance research communications Source Type: research