MOLECULAR PROPERTIES AND MODULATION OF BACTERIAL PORINS

细菌孔蛋白的分子特性和调节

• 批准号: 6510659
• 负责人: ANNE H DELCOUR
• 金额: $ 22.45万
• 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 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6510659
• 关键词: Escherichia coli; bacterial proteins; electrophysiology; membrane channels; membrane permeability; membrane potentials; mutant; polyamines; pore forming protein; protein structure function; site directed mutagenesis

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.

孔蛋白是革兰氏阴性杆菌外膜的大通道 细菌，它是亲水性溶质的主要进入途径 进入这些有机体。孔雀在传统上被认为是 毛孔永久开放，对它们的调节和作用知之甚少 行动模式。这项建议的目标是在 在分子水平上，这些蛋白的功能和调节 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。因为它们的丰富和在外部的位置 在细菌细胞表面，孔蛋白可能在宿主中发挥作用- 参与细菌感染的病原体相互作用 被证明是免疫反应的目标。孔蛋白表达的变化 在致病的大肠杆菌菌株中也观察到了这种模式 尿路感染。这个项目的意义在于 细菌外膜通透性控制的研究 这对微生物的生存起着至关重要的作用。颠覆 药物对外膜正常功能状态的影响 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，将被应用于实时测量，在分 毫秒范围内跨膜的电波波动 单一或少量渠道的活动过程。渠道 我们将在E.Coli的巨型球体或巨细胞中研究其活性， 在外膜中，部分重组为巨型脂质体。 将进行实验来研究分子机制。 主要孔蛋白的基础电压敏感性和协同性 由OmpF和OMPC基因表达。OMPC活动可以通过以下方式进行调节 膜衍生低聚糖(MDO)，果浆糖家族 在高渗透压下合成的聚合物。分子的本质 调节过程中在通道蛋白水平上发生的事件 将会被研究。我们还将确定MDO的类别，即 负责调制和定义装订位置 地点。搜索其他管制物质将在#年进行。 特定的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相关。 为了充分了解观察到的离子背后的分子机制 渠道属性，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内容相关的一般区域 具体渠道功能。最终，定点突变将 将实施以优化这些位置。这是一种 广泛应用于真核生物的结构/功能关系研究 频道。细菌系统的优点是突变 航道将在其自然环境中进行研究，而无需 注射到外来表达系统中，而孔蛋白是第一个 X-射线晶体结构已确定的通道 出版了。这些关于孔蛋白三维结构的信息 对于基因工程的设计将是极其有价值的 渠道和对数据的有意义的解释。