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性病原体，对多种抗生素具有高度耐药性。这种多药耐药是低外膜通透性和多药外排泵共同作用的结果。MexAB-OprM泵是铜绿假单胞菌的外排泵系统之一，OprM是外膜组分。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，以研究OprM的性质。结果表明，OprM是一种带门的通道蛋白。类似地，OprJ和OprN，其他外排系统的外膜成分也显示形成通道。孔蛋白作为一种外膜通道，受到了广泛的研究。孔蛋白是由β-管状体构成的，因此可以认为OprM是由β-管状体构成的。然而，CD测量令人惊讶地表明，OprM主要由α-螺旋组成。此外，还发现更多的OprJ和OprN具有相似的OprM结构。接下来，我试图确定外膜组件的膜拓扑结构，这是揭示OprM通道栅机制的必要知识。然而，确定外膜蛋白拓扑结构的常用方法远不能令人满意。然后我尝试开发一种新的方法。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。该方法的最大优点是主要适用于任何膜蛋白，例如细胞器中的膜蛋白。在这种体外方法中，通过位点定向诱变产生一系列单半胱氨酸突变蛋白。然后纯化这些蛋白质，在脂质体膜中重组，并通过膜不渗透的巯基试剂进行修饰。当引入的半胱氨酸残基和蛋白水解位点位于膜外环的同一侧时，应对消化后的蛋白产物进行标记。相反，在蛋白质水解位点的另一侧引入半胱氨酸残基的蛋白质应该显示未消化的蛋白质。然后，为了验证该方法的可用性，用该方法研究了三维结构已被分解的麦芽糖蛋白的膜拓扑结构，结果表明该方法在体外是可行的。是一种确定膜蛋白拓扑结构的有用技术。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