COLICIN CHANNELS IN VOLTAGE-CLAMPED PLANAR MEMBRANES

E1家族的粘连蛋白是一种杀菌蛋白，它能发挥其 胞质中形成电压门控离子通道的致死作用 细菌膜。水溶液中的晶体结构 一种粘菌素的状态是已知的。结肠粘连蛋白E1的形成和门控 正在研究电压钳制的、无溶剂的平面双层膜中的通道 磷脂膜。这些研究将使我们了解 在原子尺度上，蛋白质从其折叠过程中的运动 水溶性到膜结合态及其构象变化 负责渠道的开通和关闭。的属性 因此，在平面双层中的粘菌素E1通道提供了定义良好的系统 描述了生理基础上的物理化学原理 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 将蛋白质毒素(如蓖麻毒素、阿贝林毒素、白喉毒素)与载体蛋白偶联 以使毒素保持其活性并交叉靶向(例如 转化)细胞膜。例如，这就是设计时的目标 免疫毒素--与抗体偶联的毒素。这项建议是 专门针对确定的区域和残留物 当通道被电压门控时，粘菌素被转移 分解双层中这些区域的折叠图案。这个 定点突变体失活的电压依赖性 将测量在规定残基上增加或删除的电荷。这 依赖关系将给出每一个感测到的施加电压的分数 改变后的残留物。因为这个分数设置了 双分子层内的突变残基，通道的折叠模式 将会被获得。直接确认建议的折叠图案和 易位区域将通过复合膜来寻找--无谓的 亲和素，添加到跨房水隔室，与生物素化的粘连素 将生物素化残基锁定到反式侧。如果残留物是 移位，停用将被禁止。失活动力学 很大程度上依赖于跨房水隔间的pH值。酸性的 面对转室的残留物，被质子化作用中和 在低pH时(&lt；4-5)被认为是这种pH依赖性的原因。 面对反式隔间的候选酸性残基将是 突变为中性基因，以确定失活率是否 在高pH条件下增加。

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.