TRANSIENT TRIPLET ANISOTROPY INDUCED BY CAGED ATP

笼 ATP 引起的瞬态三重态各向异性

• 批准号: 3431516
• 负责人: David D Thomas
• 金额: $ 3.2万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 1989-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3431516
• 关键词: adenosine triphosphate; biological signal transduction; biophysics; calcium transporting ATPase; chemical reaction; conformation; enzyme mechanism; flash photolysis; laboratory rabbit; optical polarization; phosphorescence; stop flow technique; transducin; triplet state

Our goal is to develop and apply an instrument for observing msec transients in phosphorescence anisotropy, induced by laser photolysis of caged ATP in ATP-dependent enzymes labeled with phosphorescent dyes. These experiments will provide direct information about nanosecond and microsecond molecular dynamics coupled with enzyme action. The first stage of this project is to develop an instrument that monitors polarization anisotropy of phosphorescence continuously with high time- resolution. This requires a dual-channel detection system with two polarizers and a real-time calculation of anisotropy. In addition, in order to detect selectively the (long-lived) phosphorescence, rejecting the (short-lived) fluorescence, we will use a high-frequency pulsed laser light source (a cavity-dumped argon ion laser) and gated detection. This selectivity for phosphorescence will be augmented by using wavelength-selective interference filters. In order to obtain the high signal-to-noise necessary for msec time-resolution, we will use "structured light" techniques to focus the polarized laser beam precisely on the sample. To produce a large transient increase in the ATP concentration, the sample will be bathed in caged ATP, a photolabile ATP precursor, and the sample will be illuminated by a giant UV pulse from a XeCl excimer laser. The samples used to test and apply this new technology will be either muscle fibers, labeled with eosin-maleimide on the myosin cross-bridge, or muscle membranes, labeled with erythrosin iodoacetamide attached to the Ca-pumping ATPase. Protein rotational motions have been proposed to play important roles in these and other energy-transducing enzymes, and this technology will make possible a whole new class of biophysics experiments, in which nanosecond and microsecond molecular dynamics can be monitored directly with high sensitivity and time-resolution during the transient phase of enzymatic activity, thus helping to elucidate the molecular mechanisms of fundamental physiological processes.

我们的目标是开发和应用一种观测毫秒的仪器 激光诱导磷光各向异性的瞬态 标记的ATP依赖性酶中笼状ATP的光解 磷光染料。 这些实验将提供直接的 纳秒和微秒分子信息 动力学与酶作用相结合。 第一阶段 一个项目是开发一种监测偏振的仪器 磷光的各向异性连续与高时间- 分辨率 这需要双通道检测系统， 两个偏振器和各向异性的实时计算。 在 此外，为了选择性地检测（长寿命） 磷光，拒绝（短暂的）荧光，我们将 使用高频脉冲激光光源（腔倒空 氩离子激光器）和选通检测。 这种选择性 磷光将通过使用波长选择性增强 干涉滤光片 为了获得高信噪比， 为了获得毫秒级的时间分辨率，我们将使用“结构光” 将偏振激光束精确地聚焦在 sample. 产生ATP的大量瞬时增加 浓度，样品将沐浴在笼状ATP中， 光不稳定的ATP前体，样品将被照射 XeCl准分子激光器发出的巨大紫外线脉冲 样本用于 测试和应用这项新技术将是肌肉纤维， 在肌球蛋白横桥上用曙红-马来酰亚胺标记，或 肌膜，用赤藓红碘乙酰胺标记 附着在钙泵ATP酶上。 蛋白质旋转运动 已经被提议在这些和其他方面发挥重要作用 能量转换酶，这项技术将使 可能是一种全新的生物物理学实验， 纳秒和微秒分子动力学可以是 高灵敏度和时间分辨率直接监测 在酶活性的瞬时阶段，从而有助于 阐明基本生理学的分子机制 流程.