Protein Folding Triggered by Electron Transfer

蛋白质折叠动力学实验研究的基本要求是某种触发折叠（或展开）过程的手段。在这项研究计划中，将采用一种新的触发方法，允许在纳秒到毫秒的时间范围内进行测量。电子转移（ET）化学可以触发许多蛋白质中的折叠反应，并且有许多成熟的技术可以在短至几纳秒的时间内将电子快速注入蛋白质。具体来说，激光启动ET将用于触发三种高电位血红素蛋白的折叠；细胞色素c，细胞色素b562和细胞色素f。蛋白质折叠的复杂过程涉及超过12个数量级的时间（皮秒到分钟）的动力学。et引发的蛋白质折叠的力量在于它为直接检查打开了整个时间体系；当与蛋白质工程技术相结合时，它将允许在折叠过程的每个步骤中评估单个氨基酸的重要性。蛋白质是由20种天然存在的氨基酸组成的异聚物（多肽），通常与一种或多种辅因子结合。蛋白质的三维结构——折叠——决定了它的生物活性，它是由多肽链上的氨基酸序列决定的。因此，蛋白质折叠研究的最终目标是能够仅根据氨基酸序列预测蛋白质的三维结构。这个问题的关键在于理解将随机配置的多肽转化为完全折叠的蛋白质的一系列化学事件。有许多方法可用于研究蛋白质折叠动力学，但在大多数情况下，它们仅限于测量超过一毫秒的时间尺度。然而，有令人信服的证据表明，在许多蛋白质中，相当程度的二级和三级蛋白质结构可以在更快的时间尺度上发展。因此，需要新的实验方法来观察蛋白质折叠的最早事件。＊＊＊

9630465 Winkler The basic requirement for experimental investigations of protein-folding kinetics is some means of triggering the folding (or unfolding) process. In this research program, a new triggering method that permits measurements in the nanosecond-to-millisecond time range will be employed. Electron-transfer (ET) chemistry can trigger folding reactions in many proteins, and there are many well established techniques for rapidly injecting electrons into proteins on timescales as short as a few nanoseconds. Specifically, laser-initiated ET will be used to trigger the folding of three high-potential heme proteins; cytochrome c, cytochrome b562 and cytochrome f. The complex process of protein folding involves dynamics that span more than twelve orders of magnitude in time (picoseconds to minutes). The power of ET-initiated protein folding is that it lays open this entire time regime for direct examination; when coupled with the techniques of protein engineering, it will permit evaluation of the importance of individual amino acids in each step of the folding process. %%% Proteins are heteropolymers (polypeptides) built from the twenty naturally occurring amino acids, often in combination with one or more cofactors. The three-dimensional structure - the fold - of a protein that defines its biological activity is determined by the sequence of amino acids in the polypeptide chain. The ultimate objective in proteinfolding research then, is the ability to predict the three dimensional structure of a protein solely on the basis of its amino-acid sequence. An essential key to the problem is understanding the series of chemical events that transforms a randomly configured polypeptide into a fully folded protein. There are many methods available for studying protein-folding kinetics but, for the most part, they are limited to measurement timescales longer than one millisecond. There is compelling evidence, however, that in many proteins a substantial degre e of secondary and tertiary protein structure can develop on faster timescales. Therefore, new experimental methods are required to observe the earliest events in protein folding. ***