| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2000: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1999: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
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,
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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
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