Experimental Investigations of Protein Reconfiguration Dynamics

在寻找天然结构的过程中，多肽链是如何运动的？这些动作的时标设定了蛋白质折叠的“速度限制”。此外，骨架的局部动力学和长链片段的动力学之间的相互作用对于折叠机制是至关重要的。这个项目的目的是提供对蛋白质链重组动态的直接测量。虽然这些问题一直是密集的计算研究的主题，但目前可用的实验信息很少。实验数据的匮乏是由于技术上的困难和缺乏适当的模型系统。第一个问题最近由于快速折叠方法的发展而得到缓解。第二个问题的解决方案涉及找到在不跨越巨大的自由能障碍的情况下经历这些转变的蛋白质，这些障碍隐藏了潜在的动力学。在本项目中，一台纳秒激光诱导的温度跳跃仪将应用于蛋白质重构动力学的研究。两个互补的过程将被研究：随机疏水坍塌的动力学和导致自然拓扑的动力学。随机疏水塌陷的动力学将在一个40个残基的蛋白质中进行研究，该蛋白质最近被发现经历了随机塌陷。没有形成任何类型的特殊结构--在高温下，由于疏水作用的加强。将在折叠成稳定的熔融球体的蛋白质片段中研究塌缩到自然拓扑结构。作为时间函数的坍塌程度将通过使用荧光共振能量转移测量末端到末端的距离来确定。二级结构将通过红外吸收进行测量。这些研究的结果将是检验蛋白质折叠统计理论预测的关键。此外，它们将为计算机模拟提供重要的基准。蛋白质折叠成其功能三维结构的反应是生物学中最基本的自组织过程之一。破译蛋白质折叠的机制对于理解遗传信息如何转化为特定的生物功能以及分子进化的机制至关重要。最终，这些知识可以被用来设计“点菜”蛋白质，从而引发一场新的技术革命。蛋白质折叠反应是一个相互交织的动态和能量过程的组合。为了直接研究更微妙的动态过程，该项目建议在排除特定结构形成的特殊条件下研究蛋白质。这一策略消除了支配标准折叠反应的自由能障碍，使直接测量蛋白质的重新配置动力学成为可能。一台纳秒分辨率的激光诱导温度跳跃装置将被用来完全解决这种快速的过程。具体的目标是直接测量未折叠的蛋白质塌陷成一个随机的球体，确定蛋白质折叠的“速度限制”，并研究局部动力学和塌陷在形成天然二级结构和拓扑时的竞争。这些是蛋白质折叠中一些最基本且仍未解决的问题。

What are the motions of the polypeptide chain in its search for the native structure? The timescales for these motions set the 'speed limit' to protein folding. Moreover, the interplay between the local dynamics of the backbone and the dynamics of long chain segments is critical to the mechanisms of folding. This project aims at providing direct measurements of the dynamics of chain reconfiguration in proteins. Although these issues have been subject of intensive computational studies, very little experimental information is currently available. The scarcity of experimental data is due to technical difficulties and the lack of an appropriate model system. The first problem has been recently alleviated by the development of fast-folding methods. The solution to the second problem involves finding proteins that undergo these transitions without crossing large free energy barriers, which hide the underlying dynamics. In this project a nanosecond laser-induced temperature-jump instrument will be applied to the study of protein reconfiguration dynamics. Two complementary processes will be investigated: the dynamics of random hydrophobic collapse, and the dynamics leading to the native topology. The kinetics of random hydrophobic collapse will be studied in a 40 residue protein, which has recently been found to undergo random collapse -i.e. without formation of any kind of specific structure- at high temperature, as result of the strengthening of the hydrophobic effect. Collapse to the native topology will be investigated in a protein fragment that folds into a stable Molten Globule. The degree of collapse as a function of time will be determined by measuring the end to end distance using fluorescence resonance energy transfer. Secondary structure will be measured by infrared absorption. The results from these studies will be critical to test the predictions from the statistical theory of protein folding. Moreover, they will provide important benchmarks for computer simulations. The reactions by which proteins fold into their functional three-dimensional structures are among the most fundamental self-organization processes in biology. Deciphering the mechanisms of protein folding is critical to understand how genetic information is translated into specific biological functions, as well as the mechanics of molecular evolution. Eventually, this knowledge could be harnessed to design proteins 'a la carte', leading to a new technological revolution. Protein folding reactions are characterized by a combination of intertwined dynamic and energetic processes. To investigate directly the more subtle dynamic processes, this project proposes to study proteins in special conditions that preclude the formation of specific structures. This strategy eliminates the free energy barriers that dominate standard folding reactions, making a direct measure of the reconfiguration dynamics of proteins feasible. A laser-induced temperature jump apparatus with nanosecond resolution will be employed to resolve entirely such fast processes. The specific goals are to directly measure the collapse of unfolded proteins into a random globule, determine the 'speed limit' to protein folding, and to investigate the competition between local dynamics and collapse in forming the native secondary structure and topology. These are some of the most basic and still unresolved questions in protein folding.