Experimental Investigations of Protein Reconfiguration Dynamics

蛋白质重构动力学的实验研究

• 批准号: 0317294
• 负责人: Victor Munoz
• 金额: $ 51.47万
• 依托单位: University of Maryland, College Park
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0317294&HistoricalAwards=false
• 关键词: Experimental; Investigations; Protein; Reconfiguration; Dynamics

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.

多肽链在寻找天然结构时的运动是什么？这些运动的时间尺度为蛋白质折叠设定了“速度极限”。此外，主链的局部动力学和长链段的动力学之间的相互作用对折叠机制至关重要。该项目旨在提供蛋白质链重构动力学的直接测量。虽然这些问题一直是密集的计算研究的主题，目前只有很少的实验信息。实验数据的缺乏是由于技术困难和缺乏适当的模型系统。第一个问题最近已经通过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到找到那些经历这些转变而不跨越大的自由能势垒的蛋白质，这隐藏了潜在的动力学。本计画将利用纳秒激光诱导温度跳跃装置来研究蛋白质重组动力学。两个互补的过程将被调查：随机疏水崩溃的动力学，和动力学导致本地拓扑结构。 随机疏水性塌陷的动力学将在40个残基的蛋白质中进行研究，该蛋白质最近被发现在高温下经历随机塌陷-即不形成任何种类的特定结构，这是疏水作用增强的结果。将在折叠成稳定的熔融球的蛋白质片段中研究向天然拓扑结构的折叠。作为时间函数的塌陷程度将通过使用荧光共振能量转移测量端到端距离来确定。二级结构将通过红外吸收测量。这些研究的结果对于检验蛋白质折叠统计理论的预测至关重要。此外，它们将为计算机模拟提供重要的基准。蛋白质折叠成功能性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“按菜单”设计蛋白质，从而引发一场新的技术革命。蛋白质折叠反应的特点是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，本项目建议研究在特殊条件下排除特定结构形成的蛋白质。这种策略消除了主导标准折叠反应的自由能障碍，使蛋白质重构动力学的直接测量成为可能。一个具有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样的快速过程。具体的目标是直接测量折叠成一个随机小球的蛋白质，确定“速度限制”蛋白质折叠，并调查本地动态和崩溃之间的竞争，形成天然的二级结构和拓扑结构。这些是蛋白质折叠中一些最基本的和尚未解决的问题。