HIGH FREQUENCY EPR STUDIES OF VOLTAGE DEPENDENT ION CHANNELS

大肠杆菌素Ia是一种由626个氨基酸组成的细菌蛋白， 通过破坏主动运输和诱导离子泄漏。 已经表明，这种活性来自于以下能力： 大肠杆菌素Ia形成电压依赖性，相对非选择性 细菌质膜中的离子渗透通道。 疗效 大肠杆菌素Ia是显着的，因为一个单一的蛋白质分子在大肠杆菌素Ia中， 膜足以杀死细胞。 此外，电压 依赖的通道形成行为可以在人工 磷脂双层膜和囊泡，从而提供了理想的 离子通道物理学的一般研究系统。 高频 自旋标记的囊泡型大肠杆菌素Ia分子的EPR研究 是一种理想的手段，以解决结构细节的电压依赖 离子通道结构 概述了拟议的实验 协议如下。 一种顺磁性氮氧自由基自旋标记物， 在感兴趣的残基位置引入大肠杆菌素蛋白。 大肠杆菌素然后嵌入膜囊泡中，然后快速 与缩肽缬氨霉素混合， 潜力 此时，系统可以快速冻结以锁定 低温EPR后的蛋白构象状态 谱 或者，溶液相EPR谱可以是 得到了 在这两种情况下，氮氧化合物自旋的EPR谱 标签受环境极性的影响，因此 提供了一种方法来确定标签是在外部还是在内部（以及 也许有多深）囊泡双层。 这又导致 关于蛋白质的电压依赖性结构的信息 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.