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

Abstract

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.

Authors:
;  [1]; ;  [2];  [3];  [4];  [5];  [5];  [4];  [5];  [5];  [4];  [5];  [5];  [4];  [5];  [5];  [5];  [4];  [5] more »;  [5]; ;  [4];  [5];  [5];  [3];  [2];  [5];  [1];  [5] « less
  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)
  5. (Japan)
Publication Date:
OSTI Identifier:
22356333
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 62; Journal Issue: Pt 4; Other Information: PMCID: PMC2222563; PMID: 16582489; PUBLISHER-ID: en5154; OAI: oai:pubmedcentral.nih.gov:2222563; Copyright (c) International Union of Crystallography 2006; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United Kingdom
Language:
English
Subject:
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

Citation Formats

Tsukazaki, Tomoya, Mori, Hiroyuki, Fukai, Shuya, Numata, Tomoyuki, Perederina, Anna, Adachi, Hiroaki, Department of Electrical, Electronic and Information Engineering, Osaka University, Osaka 565-0871, CREST, JST, Saitama 332-0012, Matsumura, Hiroyoshi, CREST, JST, Saitama 332-0012, Department of Materials Chemistry, Osaka University, Osaka 565-0871, Takano, Kazufumi, CREST, JST, Saitama 332-0012, Department of Material and Life Science, Osaka University, Osaka 565-0871, Murakami, Satoshi, CREST, JST, Saitama 332-0012, PRESTO, JST, Saitama 332-0012, Institute of Scientific and Industrial Research, Osaka University, Osaka 567-0047, Inoue, Tsuyoshi, CREST, JST, Saitama 332-0012, Department of Materials Chemistry, Osaka University, Osaka 565-0871, Mori, Yusuke, Sasaki, Takatomo, Department of Electrical, Electronic and Information Engineering, Osaka University, Osaka 565-0871, CREST, JST, Saitama 332-0012, Vassylyev, Dmitry G., Nureki, Osamu, E-mail: onureki@bio.titech.ac.jp, PRESTO, JST, Saitama 332-0012, Ito, Koreaki, E-mail: onureki@bio.titech.ac.jp, and CREST, JST, Saitama 332-0012. Purification, crystallization and preliminary X-ray diffraction of SecDF, a translocon-associated membrane protein, from Thermus thermophilus. United Kingdom: N. p., 2006. Web. doi:10.1107/S1744309106007779.
Tsukazaki, Tomoya, Mori, Hiroyuki, Fukai, Shuya, Numata, Tomoyuki, Perederina, Anna, Adachi, Hiroaki, Department of Electrical, Electronic and Information Engineering, Osaka University, Osaka 565-0871, CREST, JST, Saitama 332-0012, Matsumura, Hiroyoshi, CREST, JST, Saitama 332-0012, Department of Materials Chemistry, Osaka University, Osaka 565-0871, Takano, Kazufumi, CREST, JST, Saitama 332-0012, Department of Material and Life Science, Osaka University, Osaka 565-0871, Murakami, Satoshi, CREST, JST, Saitama 332-0012, PRESTO, JST, Saitama 332-0012, Institute of Scientific and Industrial Research, Osaka University, Osaka 567-0047, Inoue, Tsuyoshi, CREST, JST, Saitama 332-0012, Department of Materials Chemistry, Osaka University, Osaka 565-0871, Mori, Yusuke, Sasaki, Takatomo, Department of Electrical, Electronic and Information Engineering, Osaka University, Osaka 565-0871, CREST, JST, Saitama 332-0012, Vassylyev, Dmitry G., Nureki, Osamu, E-mail: onureki@bio.titech.ac.jp, PRESTO, JST, Saitama 332-0012, Ito, Koreaki, E-mail: onureki@bio.titech.ac.jp, & CREST, JST, Saitama 332-0012. Purification, crystallization and preliminary X-ray diffraction of SecDF, a translocon-associated membrane protein, from Thermus thermophilus. United Kingdom. doi:10.1107/S1744309106007779.
Tsukazaki, Tomoya, Mori, Hiroyuki, Fukai, Shuya, Numata, Tomoyuki, Perederina, Anna, Adachi, Hiroaki, Department of Electrical, Electronic and Information Engineering, Osaka University, Osaka 565-0871, CREST, JST, Saitama 332-0012, Matsumura, Hiroyoshi, CREST, JST, Saitama 332-0012, Department of Materials Chemistry, Osaka University, Osaka 565-0871, Takano, Kazufumi, CREST, JST, Saitama 332-0012, Department of Material and Life Science, Osaka University, Osaka 565-0871, Murakami, Satoshi, CREST, JST, Saitama 332-0012, PRESTO, JST, Saitama 332-0012, Institute of Scientific and Industrial Research, Osaka University, Osaka 567-0047, Inoue, Tsuyoshi, CREST, JST, Saitama 332-0012, Department of Materials Chemistry, Osaka University, Osaka 565-0871, Mori, Yusuke, Sasaki, Takatomo, Department of Electrical, Electronic and Information Engineering, Osaka University, Osaka 565-0871, CREST, JST, Saitama 332-0012, Vassylyev, Dmitry G., Nureki, Osamu, E-mail: onureki@bio.titech.ac.jp, PRESTO, JST, Saitama 332-0012, Ito, Koreaki, E-mail: onureki@bio.titech.ac.jp, and CREST, JST, Saitama 332-0012. Sat . "Purification, crystallization and preliminary X-ray diffraction of SecDF, a translocon-associated membrane protein, from Thermus thermophilus". United Kingdom. doi:10.1107/S1744309106007779.
@article{osti_22356333,
title = {Purification, crystallization and preliminary X-ray diffraction of SecDF, a translocon-associated membrane protein, from Thermus thermophilus},
author = {Tsukazaki, Tomoya and Mori, Hiroyuki and Fukai, Shuya and Numata, Tomoyuki and Perederina, Anna and Adachi, Hiroaki and Department of Electrical, Electronic and Information Engineering, Osaka University, Osaka 565-0871 and CREST, JST, Saitama 332-0012 and Matsumura, Hiroyoshi and CREST, JST, Saitama 332-0012 and Department of Materials Chemistry, Osaka University, Osaka 565-0871 and Takano, Kazufumi and CREST, JST, Saitama 332-0012 and Department of Material and Life Science, Osaka University, Osaka 565-0871 and Murakami, Satoshi and CREST, JST, Saitama 332-0012 and PRESTO, JST, Saitama 332-0012 and Institute of Scientific and Industrial Research, Osaka University, Osaka 567-0047 and Inoue, Tsuyoshi and CREST, JST, Saitama 332-0012 and Department of Materials Chemistry, Osaka University, Osaka 565-0871 and Mori, Yusuke and Sasaki, Takatomo and Department of Electrical, Electronic and Information Engineering, Osaka University, Osaka 565-0871 and CREST, JST, Saitama 332-0012 and Vassylyev, Dmitry G. and Nureki, Osamu, E-mail: onureki@bio.titech.ac.jp and PRESTO, JST, Saitama 332-0012 and Ito, Koreaki, E-mail: onureki@bio.titech.ac.jp and CREST, JST, Saitama 332-0012},
abstractNote = {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.},
doi = {10.1107/S1744309106007779},
journal = {Acta Crystallographica. Section F},
number = Pt 4,
volume = 62,
place = {United Kingdom},
year = {Sat Apr 01 00:00:00 EST 2006},
month = {Sat Apr 01 00:00:00 EST 2006}
}