| Project/Area Number |
10044320
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| Research Category |
Grant-in-Aid for Scientific Research (B).
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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 | Tokai University |
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Principal Investigator |
NAKAE Taiji Tokai University School of Medicine, Professor, 医学部, 教授 (50102851)
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| Co-Investigator(Kenkyū-buntansha) |
YONEYAMA Hiroshi Tokai University School of Medicine, Assistant Professor, 医学部, 講師 (10220774)
YOSHIHARA Eisaku Tokai University School of Medicine, Associate Professor, 医学部, 助教授 (70167063)
GABRIEL Rumm University of Basel, Biozentrum, Research A
TILMAN Schir University of Basel, Biozentrum, Professor
JURG P Rosen University of Basel, Biozentrum, Professor
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1999: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1998: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Keywords | Antibiotics / Drug resistance / Efflux / Pump / Membrane protein / Crystal / X-ray diffraction / Infection / X-線結晶回析 / X線回析 / 透過性 / 耐性 |
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| Research Abstract |
Pseudomonas aeruginosa is a major pathogen in the hospital. Infection of this organism is a large problem, since this bacterium exhibits resistance to multiple species of antibiotics that is largely due to a tight outer membrane barrier and the expression of xenobiotic efflux pump. A major efflux pump in this organism is the MexAB-OprM pump. Aim of this study is to elucidate structure and function of the pump subunits. (I) The MexA subunit is an inner membrane associated protein assumed to link the MexB and OprM subunits. We tried to crystalize native from to MexA in the presence of surfactant, polyethyleneglycol 2000, LiCl and obtained 100 to 150 μmィイD13ィエD1 crystals. Preliminary X-ray diffraction analysis showed about 4 AィイD4゜ィエD4 resolution. Deacylated MexA removing fatty acid moiety by replacing N-terminal cysteine with other amino acid was also crystallized in the absence of surfactant and the current resolution is about 3.8 ィイD4゜ィエD4A. (ii) The OprM protein was purified by tagging poly-histidine extension at the carboxyl terminal end and purifying by Nickel-Sepharose column. We obtained about 100〜150 μmィイD13ィエD1 crystals in the presence of octylhydroxyethylsulfoxide, NaCl, polyethylene glycol 2000. Present resolutions is about 4.8 AィイD4゜ィエD4. Obviously more refined crystallization conditions has to be set up. (iii) The MexB structure has been studied by the gene-fusion of a reporter gene, phoA, into the C-terminal truncated MexB protein. Expression of such hybrid proteins revealed that the MexB protein bears 12 transmembrane segments leaving N- and C- termini in the cytoplasm. The MexB protein had two large periplasmic domain containing 311 and 314 amino acid residues. The MexB protein showed two-fold symmetry having highly conserved N- and C-termini halves. These properties are unique to the MexB protein.
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