COLICIN CHANNELS IN VOLTAGE-CLAMPED PLANAR MEMBRANES

电压钳位平面膜中的大肠菌素通道

• 批准号: 3307220
• 负责人: FREDRIC S COHEN
• 金额: $ 11.58万
• 依托单位: RUSH UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3307220
• 关键词: acidity /alkalinity; avidin; bacterial proteins; biotin; chimeric proteins; colicines; conformation; electrophysiology; ion transport; lipid bilayer membrane; protein transport; site directed mutagenesis; voltage gated channel

Colicins of the E1 family are bactericidal proteins which exert their lethal action by forming voltage-gated ion channels in the cytoplasmic bacterial membrane. The crystallographic structure in the water soluble state of one colicin is known. The formation and gating of colicin E1 channels are being studied in voltage-clamped, solvent-free, planar bilayer phospholipid membranes. These studies will lead to knowledge of the movements, on an atomic scale, of the protein during refolding from its water soluble to membrane-bound form and the conformational changes responsible for opening and closing of the channels. The properties of colicin E1 channels in planar bilayers thus provide a well-defined system to delineate the physico-chemical principles that underly the physiological processes of channel gating and protein translocation through bilayers. The enumeration of these principles would facilitate strategies for coupling protein toxins (e.g. ricin, abrin, diptheria) to carrier proteins so that the toxins retain their activity and cross targeted (e.g. transformed) cell membranes. This is, for example, the goal when designing immunotoxins -- toxins coupled to antibodies. This proposal is specifically directed toward determining the regions and residues of colicin that are translocated when the channel is gated by voltage and resolving the folding pattern of these regions in the bilayer. The voltage-dependence of deactivation of site-directed mutants that have charges added or deleted at defined residues will be measured. This dependence will give the fraction of the applied voltage sensed by each of the altered residues. Because this fraction sets the location of the mutated residues within the bilayer, the folding pattern of the channel will be obtained. Direct confirmation of proposed folding patterns and translocated regions will be sought by complexing membrane-impermeant avidin, added to the trans-aqueous compartment, with biotinylated colicin to lock biotinylated residues to the trans side. If the residues are translocated, deactivation will be inhibited. The deactivation kinetics are strongly dependent on the pH of the trans-aqueous compartment. Acidic residues, facing the trans compartment, that are neutralized by protonation at low pH (<4 - 5) are thought to be responsible for this pH dependence. Acidic residues that are candidates to face the trans compartment will be mutated to neutral ones to determine if the rates of deactivation are increased at high pH.

E1家族的大肠杆菌素是杀菌蛋白质，其发挥其杀菌作用。 通过在细胞质中形成电压门控离子通道的致死作用 细菌膜 水溶性的晶体结构 一种大肠杆菌素的状态是已知的。 大肠杆菌素E1的形成和门控 在电压钳位、无溶剂的平面双层膜中， 磷脂膜 这些研究将有助于了解 运动，在原子尺度上，蛋白质在重折叠过程中， 水溶性到膜结合形式和构象变化 负责打开和关闭通道。 的性质 因此，平面双层中的大肠杆菌素E1通道提供了一个明确的系统 来描述生理学的物理化学原理， 通道门控和蛋白质通过双层转运的过程。 列举这些原则将有助于制定战略， 将蛋白毒素（例如蓖麻毒素、相思豆毒素、白喉毒素）偶联到载体蛋白 使得毒素保持其活性并交叉靶向（例如， 转化的）细胞膜。 例如，这是设计时的目标， 免疫毒素--与抗体结合的毒素。 这项建议是 特别是针对确定的区域和残基， 当通道被电压门控时移位的大肠杆菌素， 解析双层中这些区域的折叠图案。 的 定点突变体失活的电压依赖性， 将测量在限定的残基处添加或删除的电荷。 这 依赖性将给出由每个传感器感测的施加电压的分数， 改变的残留物 因为这个分数设置了 双分子层内突变的残基，通道的折叠模式 将获得。 直接确认建议的折叠模式， 将通过络合膜不渗透剂来寻找易位区域， 抗生物素蛋白与生物素化大肠杆菌素一起加入到跨水隔室中 将生物素化残基锁定在反式侧。 如果残留物 移位，失活将被抑制。 失活动力学 强烈依赖于跨水隔室的pH。 酸性 通过质子化作用中和的面向反式隔室的残基 在低pH值（<4 - 5）下，被认为是造成这种pH依赖性的原因。 酸性残基是面对反式隔室的候选者， 突变为中性，以确定失活率是否 在高pH值下增加。