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法来研究小蛋白质从平衡或天然结构中被取代时结构的变化。计划中的研究利用了该实验室开发的一种新方法，该方法使用从激发态电子发色团转移的振动能量来驱动它所连接的宿主蛋白质从其天然结构到一系列部分未折叠状态，这些状态可能对应于折叠/未折叠途径中的后期中间体沿着。由于中间体结构是在有利于天然结构的溶液条件下以光学方式产生的，因此在产生未折叠的中间体之后的重折叠反应可以通过使用即时和延迟荧光方法在非常宽的时间尺度范围内、在皮秒至毫秒的范围内独立控制溶剂组成或温度来表征。锌（II）取代和无金属的细胞色素C是主要的系统进行研究。在计划的实验中，细胞色素c中的卟啉发色团作为解折叠和重折叠反应的触发器和传感器。色氨酸残基也将被用作卟啉引发的反应的遥感器。这项研究提供了一个直接测试的中心假设，蛋白质折叠迅速在生理条件下，因为他们随机移动的漏斗形势能表面，具有天然结构在漏斗的最小值。该研究还将对蛋白质结构和功能的更广泛领域产生影响，这些领域与光生物学和酶催化反应期间导致构象变化的障碍交叉过程有关。该项目在PI和co-PI的本科和研究生课程教学中发挥着不可或缺的作用。它甚至在入门课程中提供了关键的演讲主题和应用。研究参与者接触到广泛的学科，从结构生物学到化学物理学。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，该项目与一项新的外展计划有关，该计划将由PI和共同PI访问密歇根州兰辛地区的高中，为化学和物理课程提供关于激光光谱学在生物物理化学，结构生物学和相关学科中的应用的演讲。感兴趣的学生和他们的老师将被邀请参观PI，co-PI和他们的研究生，参观研究实验室，谈论科学和工程的职业生涯，也许参与研究项目。这一推广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。这一努力的目的是加强化学系在密歇根州立大学现有的努力，从代表性不足的群体招募和留住学生。