MOLECULAR PROPERTIES AND MODULATION OF BACTERIAL PORINS

孔蛋白是革兰氏阴性菌外膜的大通道 细菌，代表亲水性溶质的主要进入途径 进入这些生物体。 孔蛋白传统上被认为是 毛孔永久张开，但对其调节和作用知之甚少。 行动方式。 本提案的目标是研究 分子水平，这些蛋白质的功能和调节 大肠杆菌。 细菌是人类传染病的主要病原体之一 疾病。 由于它们的丰富性和位于外部的位置 细菌细胞表面的孔蛋白可能在宿主中发挥作用 与细菌感染相关的病原体相互作用已被证实 被证明是免疫反应的目标。 孔蛋白表达的变化 在大肠杆菌的致病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 对微生物的生存起着至关重要的作用。 颠覆 药物介导的外膜正常功能状态 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 毫秒范围内的跨膜电波动 单个或少量通道的活动过程。 频道 将在大肠杆菌的巨型原生质球或巨细胞中研究活性， 以及重组为巨型脂质体的外膜部分。 将进行实验来研究分子机制 主要孔蛋白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)，梨浆糖家族 高渗透压合成的聚合物。 分子的性质 调节过程中通道蛋白水平发生的事件 将被研究。 我们还将确定 MDO 的类别： 负责调制并定义绑定位置 网站。 将在以下时间进行其他监管物质的搜索： 促进通道开放和抑制的特殊形式分子。 其中一些最终应该与治疗方法相关。 充分了解所观察离子的分子机制 渠道属性、结构与功能之间的关系 通过使用突变通道进行探索。 各种自发 突变体将首先用于绘制与相关的一般区域 具体渠道功能。 最终，定点诱变将 实施以完善这些地点。 这是一种方法，它是 广泛应用于真核生物的结构/功能关系研究 渠道。 细菌系统的优点是突变体 渠道将在其自然环境中进行研究，而不需要 用于注射到外源表达系统中，孔蛋白是第一个 X射线晶体结构已被确定的通道 发表。 有关孔蛋白三维结构的信息 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。

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.