DETERMINING 3-D STRUCTURE OF THE PH-SENSITIVE UREA CHANNEL FROM H PYLORI

确定幽门螺杆菌 PH 敏感尿素通道的 3-D 结构

• 批准号: 8361730
• 负责人: Hans Luecke
• 金额: $ 0.55万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361730
• 关键词: 3-Dimensional; Affinity; C-terminal; Cells; Cobalt; Crystallization; Diffusion; Drops; Environment; Escherichia coli; Funding; Gastric Metaplasia; Grant; Helicobacter Infections; Helicobacter pylori; Homologous Gene; Incidence; Infection; Lipids; Mediating; National Center for Research Resources; Peptic Ulcer; Principal Investigator; Proteins; Reproduction; Research; Research Infrastructure; Resolution; Resources; Site-Directed Mutagenesis; Source; Stomach; Structure; United States National Institutes of Health; Urea; X ray diffraction analysis; X-Ray Diffraction; cancer initiation; chemical binding; cost; expression vector; malignant stomach neoplasm; periplasm; structural biology; urea transporter; vapor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. UreI from H. pylori is a pH-sensitive Urea transporter. Urea is transported to induce changes in the periplasmic space pH essential for survival and reproduction in low pH environments. Thus, H. pylori is insensitive to the acidic environment of the stomach due to UreI. About 20% of UreI-mediated infections of H. pylori result in gastric or duodenal peptic ulcer disease. Following infection there is a 20-fold increase in the incidence of gastric metaplasia and gastric cancer. UreI, and homologs, have been cloned into pET101 expression vector and expressed in E. coli BL21(43) cells. Protein was solublized using 2% DDM Protein and was purified via C-terminal His tag using a cobalt (Talon) column in the presence of E. coli polar lipid extract. Site directed mutagenesis of His residues supports that the H+-sensing ability of UreI is on the periplasmic portion opposed cytosolic. Initial crystallization conditions were obtained via hanging drop vapor diffusion at 23¿C. X-ray diffraction of crystals showed they belong to the orthorhombic space group C222 and diffract to 9¿. Since there are already some compounds that inhibit UreI, delineating a high resolution structure would enable higher affinity and selectivity of chemicals that bind to UreI. This is significant because UreI would be a prime target for eradication of gastric infection and subsequent cancer initiation.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 UreI来自H. pylori是pH敏感的尿素转运蛋白。尿素被转运以诱导周质空间pH的变化，周质空间pH对于低pH环境中的存活和繁殖至关重要。因此，H.幽门螺杆菌对UreI引起的胃的酸性环境不敏感。约20%的UreI介导的H.幽门导致胃或十二指肠消化性溃疡疾病。感染后，胃化生和胃癌的发病率增加了20倍。将UreI及其同源物克隆到pET 101表达载体中，并在E. coliBL 21（43）细胞。使用2%DDM蛋白溶解蛋白质，并在大肠杆菌存在下使用钴（Talon）柱通过C-末端His标签纯化。大肠杆菌极性脂质提取物。His残基的定点突变支持UreI的H+感应能力是在周质部分，而不是胞质。初始结晶条件通过23 ℃下的悬滴蒸气扩散获得。晶体的X射线衍射表明它们属于正交晶系空间群C222，并属于9 <$。由于已经有一些抑制UreI的化合物，因此描绘高分辨率结构将使与UreI结合的化学品具有更高的亲和力和选择性。这是重要的，因为UreI将是根除胃感染和随后的癌症启动的主要靶点。