Voltage-Gated Insertion of Colicin into Planar Bilayers

描述（由申请人提供） 关于成孔大肠杆菌素的膜输入和通道形成的研究涉及：（i）大肠杆菌素、毒素和其他膜活性蛋白质经历的大的可溶性->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，和（iv）蛋白质插入膜的途径，和（v）MW = 65，000的大肠杆菌素穿过E. coli外膜。大肠杆菌素E1通道结构域的结构，在原子分辨率解决，允许基于结构的诱变策略，以测试模型的膜结合和通道形成后的结构转变。单色氨酸和半胱氨酸突变体被用于荧光猝灭和荧光共振能量转移，以定义在膜界面层作为一个扩展的，灵活的，二维的螺旋网络中绑定的大肠杆菌素通道。与Y. N. Antonenko（莫斯科，俄罗斯）观察大肠杆菌素通道形成的动力学和相关性质。目前已开展了4个合作项目：（1）敏化染料对大肠杆菌素通道活性的光灭活作用依赖于通道结构域9号螺旋上Trp 495的存在。大肠杆菌素光失活将作为研究膜蛋白光损伤和光动力学治疗的重要模型，广泛应用于肿瘤治疗。(2)发现大肠杆菌素输入和通道形成对膜阴离子脂质含量非常敏感，并且在-60 +/-5 mV的表面电位下被“调节”。(3)纯化的外膜受体蛋白的通道活性被发现封闭外源大肠杆菌素。(4)初步实验表明，大肠杆菌素El膜结合和通道形成受到脂质界面偶极电位的影响。建议同时测量通道电流和荧光水平平面双层和电压门控大肠杆菌素插入，并在膜通道形成的动力学和途径进行分析。

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.