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性（CD）的新方法 基于差分热透镜设计。 在这个实验中 布置，CD的差异吸收特征 测量将通过光学而不是电子获得 差异程序。 这种方法将提供实质性的 CD检测灵敏度比常规传输的敏感性提高 基于仪器。 更重要的是，检测CD的能力 单个激光脉冲的时间域中的信息将允许 与要研究的瞬态现象相关的构象变化。 时间分辨CD将用于研究构象变化 与细胞色素C和 细胞色素氧化酶是距离和热力学驱动的函数 力量。 电子传输将通过激发 细胞色素C中的钌标记，以及与之相关的结构变化 细胞色素C内的电子转移，从细胞色素C到细胞色素 氧化酶从时间上解析。 时间分辨的CD技术也将是 用于研究谷霉素类似物中发生的构象变化 在插入双层时。 定位的诱变将用于 产生gramicidin衍生物，设计具有变化的形成能力 双层频道。 这些gramicidin的时间分辨CD测量 与双层互动期间的衍生物将解决这个问题 关于类似物之间的结构或动力学差异是否可以 解释它们形成渠道的不同效率。