PROTEIN DYNAMICS VIA TIME-RESOLVED CIRCULAR DICHROISM

通过时间分辨圆二色性研究蛋白质动力学

• 批准号: 3438988
• 负责人: DONALD Robert BOBBITT
• 金额: $ 9.8万
• 依托单位: UNIVERSITY OF ARKANSAS AT FAYETTEVILLE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-04-01 至 1994-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3438988
• 关键词: circular dichroism; conformation; cytochrome c; cytochrome oxidase; electron transport; gramicidin; high performance liquid chromatography; lipid bilayer membrane; membrane channels; membrane proteins; molecular dynamics; photochemistry; protein structure function; site directed mutagenesis; stop flow technique; thermodynamics

A new approach to the detection of circular dichroism (CD) is proposed based on a differential thermal lens design. In this experimental arrangement, the differential absorption characteristic of the CD measurement will be obtained via an optical, rather than an electronic differencing procedure. This approach will provide a substantial improvement in CD detection sensitivity over conventional transmission based instruments. More importantly, the ability to detect the CD information in the time domain of a single laser pulse will permit conformational changes associated with transient phenomena to be studied. Time-resolved CD will be used to study the conformational changes associated with biological electron transfer reactions in cytochrome c and cytochrome oxidase as a function of distance and thermodynamic driving force. Electron transfer will be photoinitiated by excitation of a ruthenium label in cytochrome c, and the structural changes associated with electron transfer within cytochrome c, and from cytochrome c to cytochrome oxidase resolved temporally. The time-resolved CD technique will also be used to study the conformational changes occurring in gramicidin analogs during insertion in a bilayer. Site directed mutagenesis will be used to produce gramicidin derivatives designed with an altered ability to form bilayer channels. Time-resolved CD measurements of these gramicidin derivatives during interaction with the bilayer will address the question as to whether structural or kinetic differences among the analogs can account for the different efficiencies with which they form channels.

提出了一种检测圆二色谱(CD)的新方法 基于差热透镜设计。在这个实验中 排列，光盘的差动吸收特性 测量将通过光学测量，而不是电子测量 差异化程序。这一方法将提供实质性的 CD检测灵敏度比传统传输的提高 以仪器为基础。更重要的是，能够检测CD 单个激光脉冲的时间域中的信息将允许 与瞬变现象相关的构象变化有待研究。 时间分辨CD将用于研究构象变化 与细胞色素c和细胞色素c中的生物电子转移反应有关 细胞色素氧化酶作为距离和热力学驱动的函数 武力。电子转移将通过激发一种 细胞色素c中的Ru标记及其相关的结构变化 细胞色素c内的电子转移以及从细胞色素c到细胞色素的电子转移 氧化物酶暂时分解。时间分辨CD技术也将是 用于研究GRICIDIN类似物中的构象变化 在插入双层时。定点突变将被用于 生产具有改变形成能力的革兰西丁衍生物 双层通道。这些语法修饰的时间分辨CD测量 在与双层相互作用过程中的衍生品将解决这个问题 关于类似物之间的结构或动力学差异是否可以 考虑到它们形成渠道的不同效率。