Purification, crystallization and preliminary X-ray diffraction of SecDF, a translocon-associated membrane protein, from Thermus thermophilus

Journal Article · Sat Apr 01 04:00:00 EST 2006 · Acta Crystallographica. Section F

DOI:https://doi.org/10.1107/S1744309106007779· OSTI ID:22356333

Tsukazaki, Tomoya; Mori, Hiroyuki ^[1]; Fukai, Shuya; Numata, Tomoyuki ^[2]; Perederina, Anna ^[3]; Adachi, Hiroaki ^[4]; Matsumura, Hiroyoshi ^[4]; Takano, Kazufumi ^[4]; Murakami, Satoshi ^[4]; Inoue, Tsuyoshi ^[4]; Mori, Yusuke; Sasaki, Takatomo ^[4]; Vassylyev, Dmitry G. ^[3]; Nureki, Osamu ^[2]; Ito, Koreaki ^[1]

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1. Institute for Virus Research, Kyoto University, Kyoto 606-8507 (Japan)
2. Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Kanagawa 226-8501 (Japan)
3. Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Schools of Medicine and Dentistry, Birmingham, Alabama 35294 (United States)
4. SOSHO Inc., 7-7-15-208 Asagi, Saito, Ibaraki, Osaka 567-0085 (Japan)

SecDF is a multi-path membrane protein required for efficient protein translocation and integration via translocon. Purification and crystallization of T. thermophilus SecDF have been achieved by exploiting unique crystallization techniques that allowed the collection of a 3.74 Å data set. Thermus thermophilus has a multi-path membrane protein, TSecDF, as a single-chain homologue of Escherichia coli SecD and SecF, which form a translocon-associated complex required for efficient preprotein translocation and membrane-protein integration. Here, the cloning, expression in E. coli, purification and crystallization of TSecDF are reported. Overproduced TSecDF was solubilized with dodecylmaltoside, chromatographically purified and crystallized by vapour diffusion in the presence of polyethylene glycol. The crystals yielded a maximum resolution of 4.2 Å upon X-ray irradiation, revealing that they belonged to space group P4{sub 3}2{sub 1}2. Attempts were made to improve the diffraction quality of the crystals by combinations of micro-stirring, laser-light irradiation and dehydration, which led to the eventual collection of complete data sets at 3.74 Å resolution and preliminary success in the single-wavelength anomalous dispersion analysis. These results provide information that is essential for the determination of the three-dimensional structure of this important membrane component of the protein-translocation machinery.

• OSTI ID: 22356333
• Journal Information: Acta Crystallographica. Section F, Journal Name: Acta Crystallographica. Section F Journal Issue: Pt 4 Vol. 62; ISSN ACSFCL; ISSN 1744-3091
• Country of Publication: United Kingdom
• Language: English

Related Subjects

75 CONDENSED MATTER PHYSICS
SUPERCONDUCTIVITY AND SUPERFLUIDITY
CRYSTALLIZATION
CRYSTALS
DEHYDRATION
DIFFUSION
ESCHERICHIA COLI
IRRADIATION
MEMBRANE PROTEINS
RESOLUTION
SPACE GROUPS
STIRRING
TRANSLOCATION
VISIBLE RADIATION
WAVELENGTHS
X-RAY DIFFRACTION