Structure analysis of SecA-SecYE protein translocation machinery
| Project/Area Number |
16570094
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Structural biochemistry
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| Research Institution | Kyoto University |
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Principal Investigator |
MORI Hiroyuki Kyoto Univ., Institute for Virus Res., Assistant Professor, ウイルス研究所, 助手 (10243271)
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2005: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 2004: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | Membrane protein / Protein translocation / SecA / SecYE / Thermus thermophilus / crystal structure / X-ray / X線結晶構造解析 / SecY / SecE / ATPase / チャネル |
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| Research Abstract |
In bacteria, one third of newly synthesized proteins must be translocatedacrossthecytoplasmicmembranetolocalize their final destinations. In this reaction, SecA and SecYE complex have central roles. The former is an essential ATPase to drive the reaction and the latter is an evolutionally conserved membrane protein complex, which is thought to form a specific channel for a translocatingpolypeptide. However, molecular mechanisms of the reaction still remain poorly understood. In addition, bacteria have a multi-path membrane protein, SecDF, which form a translocon-associated complex required for efficient preprotein translocation and membrane protein integration.
In this study, we established over-expression in E.coli and purification systems of these Sec factors from Thermusthemophilus HB8, and crystallized their proteins. We succeeded in determination of 3 D structure of TSecA at 2.8A resolution. Contrary to previous reports, the structure revealed occurrence of a parallel dimer. Biochemical studies also confirmed this conclusion and supported that this parallel dimer was dominant in soution. In the case of TSecDF, we obtained complete data sets at 3.7 A resolution and started to build a molecular model of TSecDF. Although we obtained good-shape TSecYE crystals, they only diffracted X-rays to 6 A. To improve the diffraction quality of TSecYE crystals, we prepared monoclonal antibodies against TSecYE and selected one that stably bound to the TSecYE complex. Fab fragments prepared from the monoclonal antibody seem to be a good tool to stabilize the TSecYE complex in crystal lattice.
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Report
(3 results)
Research Products
(9 results)
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[Journal Article] Purification, crystallization and preliminary X-ray diffraction of SecDF, a translocon-associated membrane protein, from Therm us thermophilus2006
Author(s)
Tsukazaki, T., Mori, H., Fukai, S., Numata, T., Perederina, A., Adachi.H., Matsumura, H., Takano, K., Murakami, S., Inoue, T., Mori, Y., Sasaki, T., Vassylyev, D.G., Nureki, O., Ito, K.
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Journal Title
Acta Ciystallographica Section F 62
Pages: 376-380
Description
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