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.

我们获得了以下研究结果：1。使用SF^+昆虫细胞 - 巴克洛佛病毒表达系统过表达并纯化了来自人类的多药抗性转运蛋白MDR1。纯化的MDR1在SDS-PAGE上显示单个频带。 MDR1的大肠杆菌同源物也表达并纯化。 MSBA通过7分辨率将MSBA结晶并通过X射线衍射2。大鼠的胆汁盐出口泵（BSEP）在SF^+细胞中纯化并纯化。 BSEP的一些同源物是从嗜热微生物中克隆的，并纯化以进行结晶实验。一个适合结晶工作的克隆。3。假定的非常长的链脂肪酸转运蛋白PMP70在Pichia Pastoris中表达。但是，表达水平不足以进行结晶实验。因此，我们进行了PEX19P的表征，该PEX19P参与PMP70转运到过氧化物酶体膜及其受体PEX3P.4。 KATP通道，SUR1-KIR6.2复合物在HEK293细胞中共表达。所获得的细胞显示出由其阻滞剂摄政剂激活调节的KATP通道。 SUR1-KIR6.2复合物被纯化为SDS-PAGE上的单个频带。纯化制剂的结构恶化，使用冷冻电子显微镜单个颗粒分析开始。5。 ATP是ABC转运蛋白的重要能源，但其在转运蛋白机制中的作用尚不清楚，因为ABC转运蛋白的ATP复杂结构尚未解决。因此，我们解决了与其反应中间类似物复合的萤火虫荧光素酶结构。萤火虫荧光素酶需要ATP激活底物荧光素的反应性。该结构向我们展示了反应的结构性基础，该反应如何有助于反应。