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Struchural biology of membrane protein trabsporters

膜蛋白转运蛋白的结构生物学

基本信息

批准号：
17380066
负责人：
KATO Hiroaki
金额：
$ 9.91万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

项目摘要

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.

取得了以下研究成果：1.利用Sf^+昆虫细胞-杆状病毒表达系统高效表达并纯化人多药耐药转运蛋白MDR 1。纯化的MDR 1在SDS-PAGE上显示单一条带。大肠在大肠杆菌中表达并纯化了MDR 1的同源基因MsbA。对MsbA进行了结晶和X射线衍射，分辨率为7 A.在Sf^+细胞中高效表达并纯化了大鼠胆盐输出泵（BSEP）。从嗜热微生物中克隆了BSEP的一些同源物，并纯化用于结晶实验。一个适合结晶工作的克隆.推测的极长链脂肪酸转运蛋白PMP 70在巴斯德毕赤酵母中表达。然而，表达水平不足以进行结晶实验。因此，我们对参与PMP 70转运进入过氧化物酶体膜的Pex 19 p及其受体Pex 3 p进行了表征。KATP通道SUR 1-Kir6.2复合物在HEK 293细胞中共表达。所获得的细胞显示KATP通道被其阻断剂活性调节。SUR 1-Kir6.2复合物在SDS-PAGE上纯化为单一条带。纯化后的制剂的结构测定从使用冷冻电子显微镜的单颗粒分析开始. ATP是ABC转运蛋白的重要能量来源，但由于ABC转运蛋白的ATP复合物结构尚未解决，ATP在转运机制中的作用尚不清楚。因此，我们解决了萤火虫荧光素酶结构与其反应中间体类似物复合。萤火虫荧光素酶需要ATP来激活底物荧光素的反应性。该结构揭示了该反应的结构基础，特别是ATP是如何协助该反应的。