2007 Fiscal Year Final Research Report Summary
Struchural biology of membrane protein trabsporters
| Project/Area Number |
17380066
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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 |
Applied biochemistry
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| Research Institution | Kyoto University |
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Principal Investigator |
KATO Hiroaki Kyoto University, Graduate School of Pharmaceutical Sciences, Professor (90204487)
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| Co-Investigator(Kenkyū-buntansha) |
NAKATSU Toru Kyoto University, Graduate School of Pharmaceutical Sciences, Associate Professor (50293949)
NAKANO Hiroaki Kyoto Universtiy, Graduate School of Pharmaceutical Sciences, assistant professor (10378789)
KODAN Atsushi Kyoto Universty, Graduate School of Medicine, Researcher (80360543)
MITSUOKA Kaoru National Instifute of Advanced Industrial Science and Technology, Chief Researcher (60301230)
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| Project Period (FY) |
2005 – 2007
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| Keywords | ABC transporter / P-glycoprotein / X-ray crystalloqraphy / Single Particle Analysis / membrane proteins |
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| Research Abstract |
We gained the following research results :
1. Multi-drug resistance transporter MDR1 from human was overexpressed and purified using Sf^+ insect cell-baculovirus expression system. The purified MDR1 showed a single band on SDS-PAGE. An E. coli homologue of MDR1, MsbA was also expressed and purified. MsbA was crystallized and diffracted by X-ray with 7 A resolution.
2. Bile salt export pump (BSEP) from rat was overexpressed and purified in the Sf^+ cells. Some homologues of BSEP were cloned from thermophilic microorganisms and purified for crystallization experiments. A clone suitable for the crystallization work.
3. A putative very long chain fatty acid transporter, PMP70 was expressed in Pichia pastoris. However, the expression level was not enough to do crystallization experiments. Thus, we have done characterization of Pex19p that participates PMP70 translocation into peroxisomal membrane, and its receptor Pex3p.
4. KATP channel, SUR1-Kir6.2 complex was co-expressed in HEK293 cells. The obtained cells showed KATP channel activily regulated by its blocker regents. The SUR1-Kir6.2 complex was purified as a single band on SDS-PAGE. The structure detemination of the purified preparation started by single particle analysis using cryo-electron microscopy.
5. ATP is the important energy source of ABC transporters but its role in the transporter mechanism is still unclear because ATP complex structure of ABC transporter has not solved yet. Thus, we solved the firefly luciferase structure complexed with its reaction intermediate analogue. Firefly luciferase needs ATP to activate the reactivity of the substrate, luciferin. The structure showed us structural-basis of the reaction, expecialy how ATP assists the reaction.
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