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Elucidation of the molecular structure of OprD porin bearing the protease activity and the discovery of theporin homologous with OprD

阐明具有蛋白酶活性的OprD孔蛋白的分子结构并发现与OprD同源的孔蛋白

基本信息

批准号：
09670299
负责人：
YOSHIHARA Eisaku
金额：
$ 1.98万
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：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

The recent emergence of the multidrug-resistant pathogens is going to becme a threaten for the human health. Pseudomos aeruginosa, an opportunistic pathogen possesses the resistance to a wide variety of antibiotics and this multidrug resistance is considered to be due to the concerted effects of the outer membrane barrier and the drug extrusion system.We showed that the porins in the outer membrane of P. aeruginosa form the small channels forming the higher barrier again the penetration of antibiotics through the outer membrane. Furthermore we revealed that OprD, one of porins in P.aeruginosa bears the protease activity. During the term of this project, the following studies were carried out.(1) The comparison of the amino acid sequences between OprD and serine protease lead the assumption that the catalytic residues of OprD may be Hisl56, Asp2O8 and Ser296 residues. In order to identify the catalytic residues, these amino acid were replaced with other amino acids, respectively, by usi … More ng the site-directed mutagenesis. The protease activity of these mutant proteins lowered to be 0.1% of that of native OprD.(2) When other amino acid residue (His367) besides the above ones was replaced with Gin, this mutant protein was found to eThit the similar activity with the native OprD.These results indicate clearly that QprD porin is a multi functional protein with protease activity.(3) The monoclonal antibodies against OprD showed the cross-activitywith one of the outer membrane proteins with the molecular weight of 43 kDa. This protein was revealed to be OprE3.(4) When OprE3 protein was purified, reconstituted into the liposome and ecamined by the liposome swelling method, this protein was showed to be a porin.(5) We cloned the OprE3 gene, which was denignated as opr Q gene, and determined the nucleotide sequence of this gene. When the deduced amino acid sequence of OprE3 was compared with that of OprD, OprE3 was revealed to be highly homologous with OprD.(6) In order to elucidate the three dimensinal structure of OprD protein by X ray diffraction method, we carried out the purification of OprD and protein crystallization. We have tried to grow the protein crystals under various conditions, and succeeded in growing the crystals. However, such crystals were not suitable for the x ray diffraction analysis since the crystal size was small. We are now trying to find out the best conditions for growing the large and well-ordered OprD protein crystals. Less

近年来出现的多重耐药病原菌对人类健康构成了严重威胁。铜绿假单胞菌是一种条件致病菌，对多种抗生素具有耐药性，这种多重耐药性是由外膜屏障和药物挤出系统共同作用的结果。此外，我们发现铜绿假单胞菌的孔蛋白OprD具有蛋白酶活性。在本项目期间，进行了以下研究。(1)通过与丝氨酸蛋白酶的氨基酸序列比较，假设OprD的催化残基可能是His156、Asp2O8和Ser296残基。为了鉴定催化残基，通过USI将这些氨基酸分别替换为其他氨基酸， ...更多信息进行定点诱变。这些突变蛋白的蛋白酶活性降低至天然OprD的0.1%。(2)当用Gln取代QprD孔蛋白的His367位氨基酸残基时，该突变蛋白仍具有与天然OprD相似的活性，表明QprD孔蛋白是一种具有蛋白酶活性的多功能蛋白。(3)抗OprD单克隆抗体与一种分子量为43kDa的外膜蛋白有交叉活性。该蛋白被证实是OprE3。(4)当OprE3蛋白被纯化，重组到脂质体中，并通过脂质体溶胀法进行ecamined时，该蛋白显示为孔蛋白。(5)我们克隆了Opr E 3基因，并测定了该基因的核苷酸序列，命名为opr Q基因。将推导的OprE3的氨基酸序列与OprD的氨基酸序列进行比较，发现OprE3与OprD高度同源。(6)为了用X射线衍射方法研究OprD蛋白的三维结构，我们对OprD蛋白进行了纯化和蛋白结晶。我们尝试在各种条件下生长蛋白质晶体，并成功地生长了晶体。然而，由于晶体尺寸小，这种晶体不适合于X射线衍射分析。我们现在正试图找到生长大而有序的OprD蛋白晶体的最佳条件。少