SENSITIZED PHOTOINACTIVATION OF COLICIN E1 CHANNELS

关于成孔粘菌素的研究中引起普遍兴趣的一些主要问题是：(i)粘菌素、毒素和其他膜活性蛋白所经历的大可溶性产率膜结合结构转变的性质；（ii）增强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。在原子分辨率下解决的colicin E1通道域的结构允许基于结构的诱变策略来测试膜结合和通道形成时结构转变的模型。利用单色氨酸和-Cys突变体进行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、柔性的二维螺旋网。为了开始研究从封闭通道状态到开放通道状态的结构转变，与Y. N . Antonenko（莫斯科，俄罗斯）实验室合作进行了平面脂质双分子层实验，以观察粘菌素通道形成的动力学。敏化染料存在时，大肠杆菌素通道活性被光灭活，这种作用取决于通道结构域螺旋9中Trp495的存在。在与脂质体结合的通道结构域平行实验中检测到多肽分子的交联（二聚化）。基于Antonenko实验室对革兰氏菌素阳离子选择通道的研究，本文拟探讨colicin E1通道敏化光失活的机制及相关结构变化。光失活的色氨酸依赖性允许使用单色氨酸突变体来推断参与通道形成的螺旋和特定侧链。在本研究过程中将开发的新方法将扩展Trp和cys扫描诱变的应用。初步实验还表明，黏菌素E1的膜结合和通道形成受脂质界面偶极子电位的影响。单通道和多通道测量将用于研究光失活机制和膜偶极子电位的作用。粘菌素的光失活将作为研究膜蛋白光损伤和光动力治疗的重要模型，在癌症治疗中得到广泛应用。

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.