HIGH FREQUENCY EPR STUDIES OF VOLTAGE DEPENDENT ION CHANNELS

Colicin Ia是一种626个氨基酸的细菌蛋白，可杀死细胞 通过扰乱主动运输和通过诱导离子泄漏。 已经证明，这项活动源于 Colicin Ia形成电压依赖性、相对非选择性 细菌质膜中的离子通透通道。药效 Colicin Ia的研究是值得注意的，因为在 膜足以杀死细胞。另外，电压 依赖的通道形成行为可以在人工情况下重现 磷脂双层膜和囊泡，从而提供了理想的 用于离子通道物理一般研究的系统。高频 包埋在囊泡中的自旋标记结肠素Ia分子的EPR研究 是解决电压依赖的结构细节的理想方法 离子通道结构。拟议中的实验概述 协议如下。一种顺磁性氮氧化物自旋标记物 在感兴趣的残基位置被引入到结肠素蛋白中。 然后将粘菌素植入膜泡中，然后快速 与去脂肽呋喃霉素混合建立跨膜 潜力。此时，可以快速冻结系统以锁定 蛋白质构象状态到位，随后是低温EPR 光谱学。可替换地，溶液相EPR谱可以是 获得。在任何一种情况下，氮氧化物的EPR谱自旋 标签受环境极性的影响，因此 提供一种方法来确定标签是在外面还是在里面(和 也许有多深)囊泡双层。这反过来又会导致 关于蛋白质的电压依赖结构的信息 以及它如何形成离子通道。

Colicin Ia is a 626 amino acid bacterial protein which kills cells by disrupting active transport and by inducing the leakage of ions. It has been shown that this activity derives from the ability of Colicin Ia to form voltage-dependent, relatively non-selective ion-permeable channels in the bacterial plasma membrane. The efficacy of Colicin Ia is remarkable, in that a single protein molecule in the membrane is enough to kill the cell. In addition, the voltage dependent channel forming behavior can be reproduced in artificial phospholipid bilayer membranes and vesicles, thus providing an ideal system for the general study of ion channel physics. High frequencey EPR studies of spin-labeled colicin Ia molecules imbedded in vesicles is an ideal means to address structural details of voltage-dependent ion channel structure. An overview of the proposed experimental protocol is as follows. A paramagnetic nitroxide spin label is introduced into the colicin protein at a residue position of interest. The colicin is then imbedded in membrane vesicles followed by rapid mixing with the depsipeptide valinomycin to establish a transmembrane potential. At this point the system can be rapidly frozen to lock the protein conformational state in place followed by low temperature EPR spectroscopy. Alternatively, solution phase EPR spectra may be obtained. In either case, the EPR spectrum of the nitroxide spin label is influenced by the polarity of the environment, and thus provides a means to establish if the label is outside or within (and perhaps how deep within) the vesicle bilayer. This in turn leads to information concerning the voltage-dependent structure of the protein and how it forms the ion channel.