Intramolecular Vibrational Excitation of Protein Unfolding and Refolding Dynamics

蛋白质解折叠和重折叠动力学的分子内振动激发

• 批准号: 0920101
• 负责人: Warren Beck
• 金额: $ 48.61万
• 依托单位: Michigan State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0920101&HistoricalAwards=false
• 关键词: Intramolecular; Vibrational; Excitation; Protein; Unfolding

This project employs time-resolved fluorescence spectroscopy to study how small proteins change in structure when they are displaced from the equilibrium or native structure. The planned research exploits a new method developed in this laboratory that uses the vibrational energy transferred from an excited-state electronic chromophore to drive the host protein to which it is attached from its native structure to a range of partially unfolded states that probably correspond to late intermediates along the folding/unfolding pathway. Because the intermediate structures are generated optically under the solution conditions that favor the native structure, the refolding reactions that follow the production of unfolded intermediates can be characterized with independent control of the solvent composition or temperature over a very wide range of timescales, over the picosecond to millisecond regime, through the use of prompt and delayed fluorescence methods. Zinc(II)-substituted and metal-free cytochromes c are the main systems to be studied. The porphyrin chromophore in cytochrome c serves in the planned experiments as both the trigger and the sensor for the unfolding and refolding reactions. A tryptophan residue will also be used as a remote sensor for the reactions that are triggered by the porphyrin. This research provides a direct test of the central hypothesis that proteins fold rapidly under physiological conditions because they move randomly on a funnel-shaped potential-energy surface that has the native structure at the funnel's minimum. The research will also have an impact on the broader fields of protein structure and function that relate to the barrier-crossing processes that lead to conformational changes during photobiological and enzyme-catalyzed reactions.This project plays an integral part in the teaching of undergraduate and graduate courses by the PI and co-PI. It provides key lecture topics and applications even in introductory courses. Research participants are exposed to a wide range of disciplines ranging from structural biology to chemical physics. Undergraduate students are encouraged to start working in the laboratory in the freshman year, and they usually contribute to publications by the senior year. Additionally, this project is associated with a new outreach program that will feature visits by the PI and co-PI to high schools in the Lansing, Michigan area to give presentations to Chemistry and Physics classes on the use of laser spectroscopy in biophysical chemistry, structural biology, and related disciplines. Interested students and their teachers will then be invited to visit the PI, co-PI, and their graduate students, to see the research laboratories, to talk about careers in science and engineering, and perhaps to get involved in research projects. This outreach plan will lead to interactions with students from underrepresented groups who will be recruited to undertake undergraduate studies in the sciences at Michigan State University. This effort is intended to augment an existing effort by the Department of Chemistry at MSU to recruit and retain students from underrepresented groups.

该项目采用时间分辨荧光光谱来研究微小蛋白质在偏离平衡或天然结构时结构如何变化。计划中的研究利用了该实验室开发的一种新方法，该方法利用从激发态电子发色团转移的振动能量来驱动其所附着的宿主蛋白从其天然结构进入一系列部分未折叠状态，这些状态可能对应于折叠/展开路径上的晚期中间体。由于中间体结构是在有利于天然结构的溶液条件下光学生成的，因此可以通过使用即时和延迟荧光方法，在皮秒到毫秒的范围内，在非常宽的时间尺度内独立控制溶剂组成或温度来表征产生未折叠中间体后的重折叠反应。锌(II)取代的和不含金属的细胞色素c是要研究的主要系统。细胞色素 c 中的卟啉发色团在计划的实验中充当展开和重折叠反应的触发器和传感器。色氨酸残基也将用作由卟啉触发的反应的远程传感器。这项研究提供了对中心假设的直接检验，即蛋白质在生理条件下快速折叠，因为它们在漏斗形势能表面上随机移动，而漏斗形势能表面具有漏斗最小的天然结构。该研究还将对更广泛的蛋白质结构和功能领域产生影响，这些领域涉及跨越障碍的过程，导致光生物和酶催化反应期间的构象变化。该项目在 PI 和 co-PI 的本科生和研究生课程教学中发挥着不可或缺的作用。即使在入门课程中，它也提供了关键的讲座主题和应用。研究参与者接触到从结构生物学到化学物理学的广泛学科。鼓励本科生在大一开始在实验室工作，他们通常在大四时为出版物做出贡献。此外，该项目还与一项新的外展计划相关，该计划将由 PI 和联合 PI 访问密歇根州兰辛地区的高中，向化学和物理课程介绍激光光谱在生物物理化学、结构生物学和相关学科中的应用。然后，感兴趣的学生和他们的老师将被邀请参观 PI、联合 PI 及其研究生，参观研究实验室，谈论科学和工程领域的职业，或许还可以参与研究项目。该外展计划将导致与来自代表性不足群体的学生进行互动，这些学生将被招募到密歇根州立大学进行科学本科学习。这项工作旨在加强密歇根州立大学化学系招收和留住代表性不足群体学生的现有努力。