MOLECULAR PROPERTIES AND MODULATION OF BACTERIAL PORINS

细菌孔蛋白的分子特性和调节

• 批准号: 2070154
• 负责人: ANNE H DELCOUR
• 金额: $ 10.13万
• 依托单位: UNIVERSITY OF HOUSTON
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2070154
• 关键词: Escherichia coli; X ray crystallography; bacterial proteins; bacterial toxins; gene expression; lysosomes; membrane channels; membrane permeability; membrane reconstitution /synthesis; mutant; oligosaccharides; pore forming protein; protein structure function; protoplast /spheroplast; site directed mutagenesis; voltage /patch clamp

Porins are large channels of the outer membrane of Gram-negative bacteria, which represent the major entry pathway for hydrophilic solutes into these organisms. Porins have classically been considered as permanently open pores, and little is known about their regulation and mode of action. The objectives of this proposal are to study, at the molecular level, the function and modulation of these proteins in Escherichia coli. Bacteria are one of leading agents in human infectious diseases. Because of their abundance and location at the external surface of the bacterial cells, porins may play a role in the host- pathogen interactions involved in bacterial infection and have been shown to be targets for immune responses. Changes in porin expression patterns have also been observed in virulent strains of E. coli causing urinary tract infections. The significance of this project resides in the study of the control of the bacterial outer membrane permeability which plays an vital role in microorganism's survival. The disruption of the normal functional state of the outer membrane by drug-mediated modulation of porin activity can be conceived as a potential strategy for controlling bacterial infection. The patch clamp technique, widely used in the description of eukaryotic channels, will be applied for the real-time measurement, in the sub- millisecond range, of electrical fluctuations across the membrane during the course of activity of a single or small number of channels. Channel activities will be studied in giant spheroplast or giant cells of E.coli, and in outer membrane fractions reconstituted into giant liposomes. Experiments will be conducted to investigate the molecular mechanisms underlying voltage sensitivity and cooperativity of the major porins expressed by the ompF and ompC genes. OmpC activity can be regulated by membrane-derived oligosaccharides (MDOs), a family of piroplasmic sugar polymers synthesized at high osmolarity. The nature of the molecular events taking place at the level of the channel protein during regulation will be studied. We will also determine the class of MDOs which is responsible for the modulation and define the location of the binding site. A search for other regulatory substances will be conducted, in particular form molecules which promote channel opening and inhibition. Some of these should ultimately be relevant to therapeutic approaches. To fully understand the molecular mechanisms underlying the observed ion channels properties, the relationship between structure and function will be explored by the used of mutant channels. A variety of spontaneous mutants will be used first to map the general regions relevant to specific channels functions. Ultimately, site-directed mutagenesis will be implemented to refine these locations. This is an approach which is widely used in the structure/function relationship studies of eukaryotic channels. The advantages of the bacterial system are that mutant channels are to be studied in their natural environment without the need for injection into foreign expression systems, and porins are the first channels for which an X-ray crystallographic structure has been published. This information on the three-dimensional structure of porins will be extremely valuable for the design of genetically engineered channels and the meaningful interpretation of the data.

孔是革兰氏阴性杆菌外膜的大通道， 细菌，代表亲水性溶质的主要进入途径 这些生物体。 孔蛋白通常被认为是 永久开放的毛孔，对它们的调节知之甚少， 行动方式。 这项建议的目的是研究，在 分子水平，这些蛋白质的功能和调节， 大肠埃希菌 细菌是人类感染性疾病的主要病原体之一， 疾病 由于其丰富的地理位置， 在细菌细胞的表面，孔蛋白可能在宿主中发挥作用- 病原体相互作用涉及细菌感染， 被证明是免疫反应的目标。 孔蛋白表达的变化 在E.大肠杆菌引起 尿路感染 这个项目的意义在于 控制细菌外膜通透性的研究 对微生物的生存起着至关重要的作用。 中断 外膜的正常功能状态，通过药物介导的 孔蛋白活性的调节可以被认为是一种潜在的策略， 控制细菌感染。 膜片钳技术，广泛应用于真核生物的描述， 通道，将应用于实时测量，在子- 毫秒范围内，在整个膜的电波动， 单个或少量通道的活动过程。 信道 活性将在大肠杆菌的巨球体或巨细胞中进行研究， 和外膜组分重组成巨大的脂质体。 将进行实验以研究分子机制 主要孔的潜在电压敏感性和协同性 由ompF和ompC基因表达。 OmpC活性可以通过以下方式调节： 膜源性寡糖（MDO），一个梨质糖家族 在高渗透压下合成的聚合物。 分子的本质 在调节过程中发生在通道蛋白水平的事件 将被研究。 我们还将确定MDO的类别， 负责调制和定义绑定的位置 绝佳的价钱 将对其他监管物质进行搜索， 特别是形成促进通道开放和抑制的分子。 其中一些最终应该与治疗方法有关。 为了充分理解所观察到的离子的分子机制， 通道特性，结构和功能之间的关系将 通过使用突变通道进行探索。 各种自发的 突变体将首先用于绘制与以下相关的一般区域： 具体渠道功能。 最终，定点突变将 以改善这些位置。 这是一种 广泛应用于真核生物结构/功能关系的研究 渠道 细菌系统的优点是， 通道应在其自然环境中进行研究， 用于注射到外源表达系统中，孔蛋白是第一个 通道的X射线晶体结构已被 公开. 孔蛋白的三维结构信息 对于基因工程的设计将极具价值 渠道和数据的有意义的解释。