SENSITIZED PHOTOINACTIVATION OF COLICIN E1 CHANNELS

COLICIN E1 通道的敏化光灭活

• 批准号: 6351921
• 负责人: William A. Cramer
• 金额: $ 4万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6351921
• 关键词: SDS polyacrylamide gel electrophoresis; colicines; cysteine; free radical oxygen; hydropathy; lipid bilayer membrane; membrane channels; membrane model; membrane proteins; photoactivation; photosensitizing agents; protein structure function; site directed mutagenesis; tryptophan; voltage gated channel

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.

一些主要的问题，一般感兴趣的研究孔大肠杆菌素是：（i）的性质大的可溶性产量膜结合的结构转变所经历的大肠杆菌素，毒素，和其他膜活性蛋白质;（ii）的表面结合状态，增强螺旋插入;（iii）结构变化与电压门控通道的形成。 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。 为了研究从封闭态到开放态的结构转变，我们与Y。N Antonenko（莫斯科，俄罗斯）观察大肠杆菌素通道形成的动力学。 大肠杆菌素通道活性在敏化染料的存在下被光灭活，并且这种效果依赖于通道结构域的螺旋9中Trp 495的存在。 在平行实验中检测到多肽分子的交联（二聚化），其中通道结构域结合到脂质体。 基于Antonenko实验室对短杆菌肽阳离子选择性通道的研究，建议研究大肠杆菌素E1通道的敏化光灭活机制，以及相关的结构变化。 光失活的Trp依赖性允许使用单Trp突变体来推断通道形成中的螺旋和特定侧链参与。 新的方法，将在本研究的过程中开发将扩大应用色氨酸和半胱氨酸扫描诱变。 初步实验还表明，大肠杆菌素E1膜结合和通道的形成受到脂质界面偶极电位的影响。 单通道和多通道测量将用于研究光灭活机制和膜偶极电位的作用。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。