ULTRA-FAST STUDIES OF PROTEIN FOLDING

描述：本提案的综合目标是确定 蛋白质折叠成其天然三维结构的机制 结构. 许多完全未折叠的蛋白质在被放置在 在适当的环境中，表明形成 它们的氨基酸中含有天然的三维结构 序列的 但是，确定序列如何引导 折叠未知。 为了进一步解决蛋白质折叠问题， 必须确定中间体的结构和动力学， 折叠路径 以前的工作表明，“错误折叠”状态 可以显著影响蛋白质的形成过程， 原生结构 当防止天然蛋白质的错误折叠时， 结构可以变得非常快，大约1-20 ms。 已知蛋白质表现出重折叠的“爆发阶段”， 几毫秒。 因此，如果技术 可以追踪蛋白质折叠的超快事件， 能够提供这些早期的结构信息， 中间体的 目前使用的典型混合方法（如停止流动） 由于速度太慢，无法进行这些测量，并且受到仪器的限制， 响应时间。 尽管已经进行了选定的更快的测量， 这些研究很少发表，这些研究的适用性 方法非常有限。 这里提出的是一个更一般的 现代光学方法的适用扩展，包括亚毫秒 蛋白质折叠的动力学测量。 测量利用了 研究人员实验室开发的快速混合技术， 对于光谱研究，早在启动后100毫秒， 折叠过程 具体要做的测量包括：共振 拉曼光谱法跟踪血红素轴向配位的变化; 利用酪氨酸和色氨酸残基作为报告分子的UV共振拉曼 关于它们的侧链的局部环境;荧光寿命 色氨酸侧链的研究，以获得从色氨酸到 血红素以及蛋白质大小的量度; UV-圆形 二向色性以测量α螺旋形成的程度;以及IR和拉曼 通过酰胺解卷积研究二级结构光谱 拉伸带

DESCRIPTION: The comprehensive goal of this proposal is to determine the mechanisms by which proteins fold into their native three dimensional structures. Many fully unfolded proteins spontaneously refold when placed in the proper environment indicating that the information needed to form the three- dimensional native structure is contained in their amino acid sequences. However, the rules that determine how sequence directs the folding are unknown. To further address the protein folding problem it is necessary to determine the structures and kinetics of the intermediates in the folding pathways. Previous work has indicated that "misfolded" states can significantly influence a protein's progress toward formation of the native structure. When prevented from misfolding formation of the native structure can become very fast, on the order of 1-20 ms. Further, many proteins are known to exhibit "burst phases" of refolding that occur in less than a few milliseconds. Consequently it would be of value if techniques were developed that could follow ultrafast events in protein folding and could be capable of providing structural information on these early intermediates. Typical mixing methods (eg stopped flow) currently in use are too slow to allow these measurements and are limited by instrument response times. Although selected faster measurements have been carried out there are few of these studies published and the applicability of these methods is very limited. What is proposed here is a more generally applicable expansion of modern optical methods to include submillisecond kinetics measurements of protein folding. The measurements make use of rapid mixing technology developed in the investigator's laboratory to allow for spectroscopic studies as early as 100 m sec after initiation of the folding process. The specific measurements to be done include: resonance Raman spectroscopy to follow changes in the axial coordination of the heme; UV Resonance Raman to utilize tyrosine and tryptophan residues as reporters of the local environment about their side chains; fluorescence lifetime studies of tryptophan side chains to gain a measure of distance from trp to the heme as well as a measure of the size of the protein; UV-circular dichroism to measure the extent of alpha helix formation; and IR and raman spectroscopies to study secondary structure by deconvolution of amide stretching bands.