KINETICS OF EARLY EVENTS IN PROTEIN FOLDING

描述：尽管有天文数字的可能构象，但大多数 自然进化的多肽已经获得了自发地 折叠成独特的三维结构，这就产生了一个 蛋白质的生物活性。一个详细的结构，活力和动力 一种代表性的体外可逆折叠过程的描述 一组蛋白质是了解潜在的 细胞内的蛋白质折叠及其医学意义。因为 传统动力学方法的时间分辨率有限，知之甚少 关于在亚毫秒时间尺度上折叠的过程。这些早期的 结构事件是理解蛋白质折叠方式的关键 是当前许多理论模型的发起者和主要关注点。因此， 这个项目的主要目标是直接观察形成 存在于许多蛋白质中的部分折叠的早期中间产物 在重新折叠的最初几毫秒内。一种最近开发的和 经过广泛测试的毛细混合装置使扩展 连续流动荧光和猝灭流动氢气的时间分辨 交换研究到微秒时间尺度。这种方法将是 用于研究早期结构事件的动力学，如初始结构事件 链断裂，形成稳定的氢键和长程三级键 一系列特征良好的蛋白质和一些突变体的相互作用， 用工程设计的荧光探头。其中包括线粒体和 细菌细胞色素c、泛素和葡萄球菌核酸酶，它们有 在罗德博士的实验室中被广泛地表征为常规 结构和动力学方法。结果将提供独特的洞察力 早期结构中间体的结构和动力学性质 以及它们在蛋白质折叠中的动力学作用。

DESCRIPTION: Despite an astronomical number of possible conformations, most naturally evolved polypeptides have acquired the ability to spontaneously fold into a unique three-dimensional structure, which gives rise to a protein's biological activity. A detailed structural, energetic and kinetic description of the reversible folding process in vitro for a representative set of proteins is an essential step toward understanding the underlying protein folding in the cell, and its medical implications. Because of the limited time resolution of conventional kinetic methods, little is known about the process of folding on the sub-millisecond time scale. These early structural events are the key to understanding how protein folding is initiated and are the main focus of many current theoretical models. Thus, the primary objective of this project is to directly observe the formation of partially folded early intermediates known to appear for many proteins during the first few milliseconds of refolding. A recently developed and extensively tested capillary mixing device makes it possible to extend the time resolution of continuous-flow fluorescence and quenched-flow hydrogen exchange studies into the microsecond time scale. This approach will be used to study the dynamics of early structural events, such as the initial chain collapse, formation of stable hydrogen bonds and long-range tertiary interactions for a series of well-characterized proteins and some mutants, with engineered fluorescence probes. Among these are mitochondrial and bacterial cytochromes c, ubiquitin and staphylococcal nuclease, which have been characterized extensively in Dr. Roder's laboratory by conventional structural and kinetic methods. The results will provide unique insight into the structural and dynamic properties of early structural intermediates and their kinetic role in protein folding.