title: Crystal structure analysis, overexpression and refolding behaviour of a DING protein with single mutation.
abstract: After crystallization of a certain protein-RNA complex, well diffracting crystals were obtained. However, the asymmetric unit of the crystal was too small to locate any components. Mass spectrometry and X-ray crystal structure analysis showed that it was a member of the DING protein family (HPBP). Surprisingly, the structure of HPBP reported previously was also determined accidentally as a contaminant, suggesting that HPBP has a strong tendency to crystallize. Furthermore, DING proteins were reported to relate in disease. These observations suggest that DING has potential for application in a wide range of research fields. To enable further analyses, a system for preparation of HPBP was constructed. As HPBP was expressed in insoluble form in Escherichia coli, it was unfolded chemically and refolded. Finally, a very high yield preparation method was constructed, in which 43 mg of HPBP was obtained from 1 L of culture. Furthermore, to evaluate the validity of refolding, its crystal structure was determined at 1.03 Å resolution. The determined structure was identical to the native structure, in which two disulfide bonds were recovered correctly and a phosphate ion was captured. Based on these results, it was concluded that the refolded HPBP recovers its structure correctly.
authors.affiliation: Faculty of Advanced Life Sciences, Hokkaido University, Sapporo 060-0810, Japan.
authors.forename: Zuoqi
authors.initials: Z
authors.lastname: Gai
authors.forename: Akiyoshi
authors.initials: A
authors.lastname: Nakamura
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Nagisa
authors.initials: N
authors.lastname: Hirano
authors.forename: Isao
authors.initials: I
authors.lastname: Tanaka
authors.forename: Min
authors.initials: M
authors.lastname: Yao
mesh_terms: D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D009154:Mutation; D017510:Protein Folding
chemical_list: D029968:Escherichia coli Proteins; C074432:dinG protein, E coli
keywords: DING; HPBP; crystal structure; overexpression; refolding