TIME-RESOLVED FLUORESCENCE STUDIES OF PROTEIN FOLDING

分子生物物理学的一个主要未解决的问题涉及 决定蛋白质如何折叠。 稳态荧光变化 内源性色氨酸荧光已被用于监测 蛋白质的折叠转变。 从这些 研究，只有动力学参数有关的机制折叠 可以进行过渡。 在这一建议中，一类新的 荧光实验介绍：弯曲流时间- 分辨荧光。 在这些研究中， 利用皮秒激光激发， 发射是由16个独立的时间分辨单 光子计数通道 以这种方式，完整的时间分辨 可以在短至10毫秒内获得荧光衰减。 时间分辨实验的附加信息量 允许停流研究不仅检查 转型，也是机制。 研究是在 含有单一色氨酸的葡萄球菌核酸酶和位点- 特定的突变体 内源性色氨酸荧光的变化 1971年，Anfinsen的研究小组首次使用这种蛋白质， 明确证明存在动力学中间体 蛋白质的重折叠。 然而，没有洞察到 该蛋白质的中间形式的物理性质具有 曾经获得过。 从毫秒级变化的检验中 在核酸酶的寿命和旋转相关时间中， 再折叠，局部化和类天然结构的时间过程 形成，以及溶剂暴露的历史， 单个色氨酸残基。 一系列位点特异性突变体 专门设计用来 单色氨酸与受体间的转移实验 位于整个核酸酶结构中的基团。 从 检查这些突变体毫秒电影的 获得了重折叠转变。 虽然葡萄球菌 核酸酶及其位点特异性突变体被靶向于 调查，本提案中制定的方法将 有利于所有利用变化动力学的研究 在稳态荧光监测生物转变 (e.g.,配体结合，蛋白质缔合反应，蛋白质：DNA 互动等）。

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.).