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

铜绿假单胞菌是一种机会致病菌，对多种抗生素表现出高度的耐药性。这种多药耐药性可归因于低外膜通透性和多药外排泵的协同作用。MexAB-OprM泵是铜绿假单胞菌的外排泵系统之一，而OprM是外膜组件。本研究的主要目的是揭示OprM功能的分子机制。首先，我纯化了OprM，并在脂质体膜中重组，研究了OprM的性质。结果表明，OprM是一种带有门的通道蛋白。类似地，OprJ和OprN，其他外排系统的外膜成分也被显示为形成通道。作为一种外膜通道，孔蛋白受到了广泛的研究。波林是由β-Barrel构成的，因此可以合理地假设Oprm是由β-Barrel构成的。然而，CD测量令人惊讶地发现，OprM主要由α-螺旋组成。此外，…更多的OprJ和OprN也被发现与OprM具有相似的结构。接下来，我试图确定外膜组件的膜拓扑，这是揭示OprM通道的门机制的必要知识。然而，常用的确定外膜蛋白拓扑结构的方法远远不能令人满意。然后我试着开发了一种新的方法。因此，我能够设计出与通常的体内方法完全不同的体外方法。最大的优点是，这种方法主要适用于任何膜蛋白，例如细胞器中的蛋白。在这种体外方法中，通过定点突变产生了一系列单一的半胱氨酸突变蛋白。然后对这些蛋白质进行纯化，在脂质体中重组，并用膜不透性的巯基试剂进行修饰。当引入的半胱氨酸残基和蛋白水解点位于膜外环的同一侧时，消化的蛋白产物应该被标记。反之，半胱氨酸残基位于蛋白水解区的另一侧的蛋白质将呈现未被消化的蛋白质的标记。然后，为了验证这种新方法的可用性，用这种方法研究了已经解析了三维结构的麦芽糖蛋白的膜拓扑结构，结果表明这种体外方法是可行的。是一种确定膜蛋白拓扑结构的有用技术。较少

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