Voltage-Gated Insertion of Colicin into Planar Bilayers

描述(由申请人提供) 关于成孔性粘连蛋白的膜输入和通道形成的研究涉及：(I)由粘连蛋白、毒素和其他膜活性蛋白经历的大的可溶性--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，以及(Iv)蛋白质插入膜的途径，以及(V)分子量为65,000的粘连蛋白跨大肠杆菌外膜转运的机制。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。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，以观察粘连素通道形成的动力学和相关性质。有4个合作项目：(1)在敏化染料存在的情况下，Colicin通道活性被光灭活，这一效应依赖于通道结构域9螺旋上Trp495的存在。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。(2)Colicin的输入和通道形成对膜阴离子脂类含量非常敏感，并在-60+/-5 mV的表面电位下“调谐”。(3)纯化的外膜受体蛋白的通道活性被外源性粘菌素阻断。(4)初步实验表明，结肠素EL膜结合和通道形成受脂界面偶极子电位的影响。建议用水平平面双层膜同时测量通道电流和荧光，并分析电压门控粘连素插入膜和膜内通道形成的动力学和途径。

DESCRIPTION (provided by applicant) Studies on membrane import and channel formation of the pore-forming colicins concern: the nature of (i) the large soluble --> membrane-bound structural transition undergone by colicins, toxins, and other membrane-active proteins, (ii) the surface-bound state that potentates helix insertion, (iii) structure changes associated with voltage-gated channel formation, and (iv) the pathway of protein insertion into the membrane, and (v) the mechanism by which the MW = 65,000 colicins are translocated across the E. coli outer membrane. 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. 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 and related properties. There have been 4 collaborative projects: (1) 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. Colicin photoinactivation will serve as an important model for study of photodamage of membrane proteins and photodynamic therapy, widely used in cancer treatment. (2) Colicin import and channel formation was found to be very sensitive to membrane anionic lipid content and to be "tuned" at a surface potential of -60 +/- 5 mV. (3) The channel activities of purified outer membrane receptors proteins were found to be occluded by exogenous colicin. (4) Preliminary experiments indicate that colicin El membrane-binding and channel formation is affected by the lipid interfacial dipole potential. It is proposed to simultaneously measure channel current and fluorescence with horizontal planar bilayers and to analyze the kinetics and pathway of voltage-gated colicin insertion into, and channel formation in, the membrane.