The outer membrane components of xenobiotic efflux pumps are discovered to be members of a novel channel family with the unique structure

外源物质外排泵的外膜成分被发现是具有独特结构的新型通道家族的成员

基本信息

批准号：
11670275
负责人：
YOSHIHARA Eisaku
金额：
$ 2.24万
依托单位：
Tokai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

Pseudomonas aeruginosa is an opportunistic pathogen and exhibits highly resistance to a wide variety of antibiotics. Such multidrug resistance is attributable to the synergy of the low outer membrane permeability and the multidrug efflux pump. MexAB-OprM pump is one of the efflux pump systems in P.aeruginosa and OprM is an outer membrane component. The main purpose of this research is to reveal the molecular mechanism of the OprM function. First I purified OprM and reconstituted in liposome membranes to investigate the properties of OprM.Consequently it was demonstrated that OprM is a channel protein with gate. Similarly OprJ and OprN, an outer membrane component of other efflux systems were also shown to form a channel. As an outer membrane channel, porin has been intensively studied. Porin is made up form β-barrel and so it was reasonable to assume that OprM is constructed by β-barrel. However, the CD measurement surprisingly showed that OprM is made mainly from α-helix. Furthermore, … More OprJ and OprN were also found to share a similar structure of OprM.Next, I tried to determine the membrane topology of the outer membrane component, which is an essential knowledge to reveal the gate mechanism of OprM channel. However, the usual methods to determine the topology of the outer membrane proteins are far from satisfaction. Then I made a try to develop a novel method. Consequently I was able to design the in vitro method that is totally different from the usual in vivo methods. It is the most advantage that this method is principally applicable to any membrane proteins, e.g. those in the organelles. In this in vitro method, a series of single cystein mutant proteins is created by site directed mutagenesis. Then these proteins are purified, reconstituted in liposome membranes and subjected to the modification by the membrane-impermeable sulfhydryl reagent. When the introduced cystein residue and proteolytic site are located in the same side of extramembranous loop, the digested protein product is supposed to be labelled. On the contrary, the protein with the cystein residue introduced in the opposite side to the proteolytic site is supposed to exhibit the undigested protein with the label.Then, to test the usability of this novel method, the membrane topology of maltoporin whose three dimensional structure has been resolved was studied by using this method Consequently it was indicated that this in vitro method. is a useful technique to determine the topology of membrane proteins. Less

铜绿假单胞菌是一种机会性病原体，对多种抗生素表现出高度耐药性，这种多药耐药性归因于低外膜渗透性和多药外排泵的协同作用。铜绿假单胞菌和OprM是一种外膜成分。本研究的主要目的是首先揭示OprM功能的分子机制。我纯化了 OprM 并在脂质体膜中重构，以类似地研究 OprM 的特性。结果证明，OprM 是一种具有门的通道蛋白，OprJ 和 OprN（其他外排系统的外膜成分）也被证明可以形成通道。作为外膜通道，孔蛋白是由 β 桶组成的，因此可以合理地假设 OprM 是由 β 桶构成的。令人惊讶的是，OprM 主要由 α 螺旋组成。此外，OprJ 和 OprN 也被发现具有与 OprM 相似的结构。接下来，我尝试确定外膜成分的膜拓扑结构，这是一个重要的知识。然而，确定外膜蛋白拓扑结构的常用方法已经得到满足，然后我尝试开发一种新的方法，即体外方法。完全不同与通常的体内方法不同，该方法主要适用于任何膜蛋白，例如细胞器中的膜蛋白。在该体外方法中，通过定点诱变产生一系列单半胱氨酸突变蛋白。当引入的半胱氨酸残基和蛋白水解位点位于脂质体膜的同一侧时，这些蛋白质被纯化、重构在脂质体膜中并通过膜不可渗透的巯基试剂进行修饰。膜外环，消化的蛋白质产物应该被标记，相反，在蛋白水解位点的另一侧引入半胱氨酸残基的蛋白质应该显示带有标记的未消化的蛋白质。利用该方法对已解析三维结构的麦芽孔蛋白的膜拓扑结构进行了研究，表明该体外方法是一种可用于确定膜蛋白拓扑结构的技术。