SENSITIZED PHOTOINACTIVATION OF COLICIN E1 CHANNELS

在对成孔粘附素的研究中，人们普遍感兴趣的一些主要问题是：(I)由粘连素、毒素和其他膜活性蛋白经历的膜结合结构转变的性质；(Ii)促进螺旋插入的表面结合状态；(Iii)与电压门控通道形成相关的结构变化。在原子分辨率下求解的colicin E1通道域的结构，允许基于结构的突变策略测试膜结合和通道形成时的结构转换模型。单色氨酸和半胱氨酸突变体用于荧光猝灭和荧光共振能量转移，将结合在膜界面层的粘菌素通道定义为一个延伸的、灵活的二维螺旋网络。为了启动从闭合到开放通道状态的结构转变的研究，我们与俄罗斯莫斯科的Y.N Antonenko实验室合作，进行了平面脂质双层实验，以观察粘连素通道的形成动力学。在敏化染料存在的情况下，Colicin通道活性被光灭活，这种作用依赖于通道结构域9螺旋上Trp495的存在。在与脂质体结合的通道结构域的平行实验中检测到多肽分子的交联性(二聚化)。基于Antonenko实验室对粘杆菌素阳离子选择性通道的研究，我们建议研究粘菌素E1通道的敏化光失活机制及其相关的结构变化。光失活的Trp依赖性允许使用单个Trp突变体来推断参与通道形成的螺旋和特定的侧链。在这项研究过程中将开发的新方法将扩大Trp和Cys扫描突变的应用。初步实验还表明，结肠素E1膜结合和通道形成受脂质界面偶极电势的影响。单通道和多通道测量都将被用来研究光灭活的机制和膜偶极子电位的作用。Colicin光失活将成为研究膜蛋白光损伤和光动力疗法的重要模型，被广泛应用于癌症治疗。

Some major problems of general interest from studies on the pore-forming colicins are: the nature of (i) the large soluble yields membrane-bound structural transition undergone by colicins, toxins, and other membrane-active proteins; (ii) the surface-bound state that potentiates helix insertion; (iii) structure changes associated with voltage-gated channel formation. The structure of the colicin E1 channel domain, solved at atomic resolution, allows structure-based mutagenesis strategies to test models for structural transitions upon membrane-binding and channel formation. Single-Trp and -Cys mutants were used in fluorescence quenching and fluorescence resonance energy transfer to define the colicin channel bound in the membrane interfacial layer as an extended, flexible, two-dimensional helical net. To initiate studies on the structure transition from closed- to open-channel state, planar lipid bilayer experiments have been carried out in collaboration with the lab of Y. N Antonenko (Moscow, Russia) to observe the kinetics of colicin channel formation. Colicin channel activity was photoinactivated in the presence of sensitizing dyes, and this effect depended on the presence of Trp495 in helix 9 of channel domain. Cross-linking of the polypeptide molecule (dimerization) was detected in parallel experiments with channel domain bound to liposomes. Based on studies of the Antonenko lab on the gramicidin cation-selective channel, it is proposed to investigate the mechanism of sensitized photoinactivation of the colicin E1 channel, and associated structure changes. The Trp-dependence of photoinactivation allows the use of single-Trp mutants to infer the helices and specific side chain involvement in channel formation. New approaches that will be developed in the course of this study will extend the application of Trp- and Cys-scanning mutagenesis. Preliminary experiments show also that colicin E1 membrane-binding and channel formation is affected by the lipid interfacial dipole potential. Both single-and multi- channel measurements will be used to investigate the mechanism photoinactivation and the role of the membrane dipole potential. Colicin photoinactivation will serve as an important model for study of photodamage of membrane proteins and photodynamic therapy, widely used in cancer treatment.