SENSITIZED PHOTOINACTIVATION OF COLICIN E1 CHANNELS

COLICIN E1 通道的敏化光灭活

• 批准号: 6499507
• 负责人: William A. Cramer
• 金额: $ 3.58万
• 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/6499507
• 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.

对孔形成的大肠菌素的研究的总体兴趣的一些主要问题是：（i）大溶解性产生膜结构的结构过渡，由结肠蛋白，毒素和其他膜活性蛋白进行； （ii）增强螺旋插入的表面结合状态； （iii）与电源门控通道形成相关的结构变化。 在原子分辨率上求解的结菌素E1通道结构域的结构允许基于结构的诱变策略测试模型在膜结合和通道形成时的结构过渡。单trp和-cys突变体用于荧光淬灭和荧光共振能传递，以定义膜界面层中结合的结肠蛋白通道作为扩展，柔性，二维螺旋网。 为了启动从闭合通道状态到开放通道状态的结构过渡的研究，已经与Y. N Antonenko（俄罗斯莫斯科）的实验室合作进行了平面脂质双层实验，以观察结肠通道形成的动力学。 在敏化染料的存在下，将结菌素通道活性在光中激活，并且这种作用取决于通道域的螺旋9中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（二聚化）的交联。 基于Gramicidin阳离子选择通道上的Antonenko实验室的研究，提议研究结菌素E1通道的敏化光激活的机理，以及相关的结构变化。 光激活的TRP依赖性允许使用单TRP突变体来推断螺旋和特定的侧链参与通道形成。 在这项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，结菌素E1膜结合和通道的形成受脂质界面偶极电位的影响。 单个和多通道测量值将用于研究机理光激活和膜偶极电位的作用。结菌素光活化将作为研究膜蛋白和光动力疗法的光损伤的重要模型，该模型广泛用于癌症治疗。