Spectroscopic Studies of Protein Folding

这项工作的主要目的是收集蛋白质折叠初始阶段的动力学和结构信息。利用激光闪光光解和改进的快速混合技术来启动折叠反应，将动力学折叠研究的时间分辨率扩展到亚毫秒时间范围。由此产生的构象变化将由瞬态光谱在可见光和近紫外区域，以及稳态荧光和皮秒时间分辨荧光监测。这些技术将应用于细胞色素c和通过定点诱变制备的变异。利用共价连接的血红素基团的氧化还原和配体结合特性，可以通过光还原或光解与未折叠蛋白质中还原血红素铁结合的一氧化碳配体来启动细胞色素c的折叠反应。多肽链的崩溃将通过时间分辨荧光与快速混合技术相结合进行监测，最初使用连续流，后来使用喷射混合装置进行亚毫秒时间尺度的测量。多肽链是如何自发折叠成具有生物活性的蛋白质的问题，在整个分子生物学中具有基础和实际的重要性，但我们才刚刚开始揭示这种非凡反应的一些原理。由于传统动力学技术的时间分辨率有限，目前还没有在亚毫秒时间尺度上对蛋白质折叠事件进行直接的实验观察。然而，这些快速的结构事件是理解蛋白质折叠如何启动的关键，这是许多解决蛋白质折叠问题的理论努力的焦点。涉及新型光学触发和快速混合方法的动力学研究将为蛋白质折叠早期阶段的链崩溃和二次形成提供独特的见解。＊＊＊

9306367 Roder The main objective of the proposed work is to gather kinetic and structural information on the initial stages of protein folding. The time resolution of kinetic folding studies will be extended into the sub-millisecond time range by using laser flash photolysis and improved rapid mixing techniques to initiate the folding reaction. The resulting conformational changes will be monitored by transient spectroscopy in the visible and near-UV region, as well as steady-state fluorescence and picosecond time-resolved fluorescence. These techniques will be applied to cytochrome c and variants prepared by site-directed mutagenesis. By taking advantage of the redox and ligand binding properties of the covalently attached heme group, it is possible to initiate the folding reaction of cytochrome c by photoreduction, or by photodissociation of a carbon monoxide ligand bound to the reduced heme iron in the unfolded protein. The collapse of the polypeptide chain will be monitored by time-resolved fluorescence in conjunction with rapid mixing techniques, initially using continuous-flow and later a jet mixing device for measurements on a sub-millisecond time scale. %%% The question of how a polypeptide chain can spontaneously fold into a biologically active protein is of fundamental and practical importance throughout molecular biology, but we are only just beginning to unravel some of the principles of this remarkable reaction. Because of the limited time resolution of conventional kinetic techniques, there are at present no direct experimental observations on protein folding events on the sub- millisecond time scale. Yet, these rapid structural events are key to understanding how protein folding is initiated, which is the focus of numerous theoretical efforts to solve the protein folding problem. Kinetic studies involving novel optical triggering and fast mixing methods will provide unique insight into chain collapse and secondary formation during early stages of protein folding. ***