TIME-RESOLVED FLUORESCENCE STUDIES OF PROTEIN FOLDING

分子生物物理学尚未解决的主要问题之一是 决定蛋白质如何折叠。稳态荧光变化 利用本征色氨酸荧光来监测 蛋白质多年来的折叠转变。从这些 研究，只有关于折叠机制的动力学争论 转型是可以实现的。在这项提案中，一类新的 介绍了荧光实验：弯流时间-时间曲线。 分辨荧光。在这些研究中，重复性很高 利用皮秒激光进行激发，荧光 发射由16个独立的时间分辨信号检测 光子计数通道。以这种方式，完全的时间分辨 荧光衰减可以在短短10毫秒内获得。 时间分辨实验的附加信息量 允许停流研究不仅检查 不仅是过渡，更是机制。研究是在 含有葡萄球菌核酸酶和位点的单一色氨酸- 特定的突变体。内源性色氨酸荧光的变化 安芬森的研究小组于1971年首次使用这种蛋白质 明确证明动力学中间体的存在 在蛋白质的重折叠中。然而，没有对 这种蛋白质的物理性质中间体(S)有 从未被获得过。从对毫秒级变化的检查 核酸酶的寿命和旋转相关时间 再褶皱--局部化类原生构造的时程 观察到的形成，以及溶剂暴露历史 单一的色氨酸残留物。一系列定点突变体 都是专门为执行能量而设计的 单一色氨酸与受体之间的转移实验 基团分布在整个核酸酶结构中。从… 检查这些突变体的毫秒级电影 得到了重折叠跃迁。虽然金黄色葡萄球菌 核酸酶及其定点突变体的靶标是 在调查中，本提案中制定的方法将 对所有利用变化动力学的研究都是有益的 在稳态荧光中监测生物转变 (例如，配体结合、蛋白质缔合反应、蛋白质：DNA 交互等)。

One of the major unsolved problems of molecular biophysics involves determining how proteins fold. Steady-state fluorescence changes of intrinsic tryptophan fluorescence have been utilized to monitor folding transitions in proteins for many years. From these studies, only kinetic arguments concerning the mechanism of folding transitions could be made. In this proposal, a new class of fluorescence experiments are introduced: stooped-flow time- resolved fluorescence. In these studies, a high repetition picosecond laser is utilized for excitation, and the fluorescence emission is detected by sixteen independent time-resolved single photon counting channels. In this manner, complete time-resolved fluorescence decay can be obtained in as little as 10 milliseconds. The addition information content of the time-resolved experiments allows stopped-flow studies to examine not just the kinetics of a transition but also the mechanism. Studies are performed on the single tryptophan containing Staphylococcal Nuclease and site- specific mutants. Changes in the intrinsic tryptophan fluorescence in this protein was first used by Anfinsen's group in 1971 to unequivocally demonstrate the existence of kinetic intermediates in the refolding of proteins. However, no insight into the physical nature of the intermediate form(s) of this protein have ever been obtained. From examination of the millisecond changes in lifetimes and rotational correlation times of nuclease during refolding, the time-course of localized and native-like structure formation are observed, as well as the solvent exposure history of the single tryptophan residue. A series of site-specific mutants have been constructed, specifically designed for performing energy transfer experiments between the single tryptophan and acceptor groups located throughout the structure of nuclease. From examination of these mutants millisecond motion pictures of the refolding transition are obtained. Although Staphylococcal Nuclease and its site-specific mutants are targeted for investigation, the methodologies developed in this proposal will be beneficial for all studies which utilize the kinetics of changes in steady-state fluorescence to monitor biological transitions (e.g., ligand binding, protein association reactions, protein:DNA interactions, etc.).