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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}
}
  • The pyrimidine nucleoside phosphorylase TTHA1771 from T. thermophilus HB8 has been overexpressed, purified and crystallized. The crystals diffract X-rays to 1.8 Å resolution using synchrotron radiation. Pyrimidine nucleoside phosphorylase (PYNP) catalyzes the reversible phosphorolysis of pyrimidines in the nucleotide-synthesis salvage pathway. In order to study the structure–thermostability relationship of this enzyme, PYNP from the extreme thermophile Thermus thermophilus HB8 (TTHA1771) has been cloned, overexpressed and purified. The TTHA1771 protein was crystallized at 291 K using the oil-microbatch method with PEG 4000 as a precipitant. A native data set was collected to 1.8 Å resolution using synchrotron radiation. The crystal belongsmore » to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 58.83, b = 76.23, c = 103.86 Å, β = 91.3°.« less
  • The crystallization and preliminary X-ray diffraction analysis of the l-fuculose-1-phosphate aldolase (FucA) from T. thermophilus HB8. Native diffraction data set was collected to a resolution of 1.9 Å. Fuculose phosphate aldolase catalyzes the reversible cleavage of l-fuculose-1-phosphate to dihydroxyacetone phosphate and l-lactaldehyde. The protein from Thermus thermophilus HB8 is a biological tetramer with a subunit molecular weight of 21 591 Da. Purified FucA has been crystallized using sitting-drop vapour-diffusion and microbatch techniques at 293 K. The crystals belong to space group P4, with unit-cell parameters a = b = 100.94, c = 45.87 Å. The presence of a dimer ofmore » the enzyme in the asymmetric unit was estimated to give a Matthews coefficient (V{sub M}) of 2.7 Å{sup 3} Da{sup −1} and a solvent content of 54.2%(v/v). Three-wavelength diffraction MAD data were collected to 2.3 Å from zinc-containing crystals. Native diffraction data to 1.9 Å resolution have been collected using synchrotron radiation at SPring-8.« less
  • To elucidate the mechanism of regulation of aspartate kinase, the regulatory subunit (the β subunit of T. thermophilus aspartate kinase) was crystallized in the presence of the inhibitor threonine. Aspartate kinase (AK) from Thermus thermophilus, which catalyzes the first step of threonine and methionine biosynthesis, is regulated via feedback inhibition by the end product threonine. To elucidate the mechanism of regulation of AK, the regulatory subunit (the β subunit of T. thermophilus AK) was crystallized in the presence of the inhibitor threonine. Diffraction data were collected to 2.15 Å at a synchrotron source. The crystal belongs to the cubic spacemore » group P4{sub 3}32 or P4{sub 1}32, with unit-cell parameters a = b = c = 141.8 Å.« less
  • The molybdopterin synthase from T. thermophilus HB8 was cloned, expressed, purified and crystallized. The crystals belong to space group P2{sub 1} and diffracted to a resolution of 1.64 Å. Thermus thermophilus is a Gram-negative aerobic thermophilic eubacterium which can grow at temperatures ranging from 323 to 355 K. In addition to their importance in thermostability or adaptation strategies for survival at high temperatures, the thermostable enzymes in thermophilic organisms contribute to a wide range of biotechnological applications. The molybdenum cofactor in all three kingdoms consists of a tricyclic pyranopterin termed molybdopterin that bears the cis-dithiolene group responsible for molybdenum ligation.more » The crystals of molybdopterin synthase from T. thermophilus HB8 belong to the primitive monoclinic space group P2{sub 1}, with unit-cell parameters a = 33.94, b = 103.32, c = 59.59 Å, β = 101.3°. Preliminary studies and molecular-replacement calculations reveal the presence of three monomers in the asymmetric unit.« less
  • The putative fumarylacetoacetase TTHA0809 from T. thermophilus HB8 has been overexpressed, purified and crystallized. The crystals diffracted X-rays to 2.2 Å resolution using synchrotron radiation. Fumarylacetoacetase catalyzes the final step of tyrosine and phenylalanine catabolism. A recombinant form of the fumarylacetoacetase family member TTHA0809 from Thermus thermophilus HB8 has been crystallized by the oil-microbatch method using sodium chloride as a precipitating agent. The crystals belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 93.3, b = 73.4, c = 122.6 Å, β = 111.8°. The crystals are most likely to contain two dimers in the asymmetricmore » unit, with a V{sub M} value of 3.32 Å{sup 3} Da{sup −1}. Diffraction data were collected at 2.2 Å resolution using synchrotron radiation at beamline BL26B1 of SPring-8, Japan.« less