RUI: Molecular Mechanisms of Short-Range Electron Transfer in Metalloproteins

该项目将重点了解蛋白质中短程电子转移的分子水平机制。所有生命系统都通过占据高能态的电子获取能量，或者通过呼吸链（食物），或者通过光（光合作用）。电子转移是许多负责储存、转移和转化这种能量的蛋白质的重要​​功能。虽然人们对控制长距离电荷转移的因素有一些了解，但对短于 1 nm 的距离的生物电子转移知之甚少。填补这一空白将增进生物能量学基本步骤的知识。通过开发计算和实验模型来预测和解释这些反应的关键参数，将获得对短程生物电子转移的统一理解。此外，该项目还将为本科生提供研究培训，并支持科学课程的创新。将要研究的系统是PpcA，一种来自硫还原地杆菌的3血红素细胞色素，经过基因改造并用多种光敏剂共价标记，将用作研究电荷转移反应的模型系统。将在室温和低温下通过时间分辨荧光和吸光度光谱研究反应动力学。蛋白质-光敏剂复合物的结构完整性将通过 SAXS 和 CD 光谱进行监测。将进行广泛的全原子分子动力学模拟来预测结构并评估结构动力学。这些预测将通过多种结构技术进行实验测试。收集的动力学数据将在分子结构的背景下进行分析，并将用于测试和修改当前可用的用于预测电子转移速率和路径的计算和理论方法。该项目还将促进詹姆斯麦迪逊大学许多高级实验室课程的课程更新。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

This project will focus on understanding the molecular level mechanism of short-range electron transfer in proteins. All living systems obtain energy through electrons occupying high energy states, either through respiratory chains (food) or from light (photosynthesis). Electron transfer is a vital function of the many proteins responsible for storage, transfer, and transformation of this energy. While there is some understanding of the factors controlling charge transfer over longer distances, little is known about biological electron transfer at distances shorter than 1 nm. Filling this gap will advance the knowledge of the fundamental steps in bioenergetics. By developing computational and experimental models to predict and explain the key parameters of these reactions, a cohesive understanding of short-range biological electron transfer will be obtained. In addition, the project will provide research training for undergraduates and will support innovations in the science curriculum.The system that will be studied is PpcA, a 3 heme cytochrome from Geobacter sulfurreducens, genetically modified and covalently labeled with a number of photosensitizers will be used as a model system to study charge transfer reactions. The kinetics of the reactions will be studied with time-resolved fluorescence and absorbance spectroscopies at room and cryogenic temperatures. The structural integrity of protein-photosensitizer complexes will be monitored with SAXS and CD spectroscopy. Extensive all-atom molecular dynamics simulations will be performed to predict structures and to evaluate structural dynamics. These predictions will be tested experimentally with a number of structural techniques. The collected kinetic data will be analyzed in the context of molecular structures and will be used to test and revise currently available computational and theoretical approaches for the prediction of electron transfer rates and pathways. This project will also facilitate curricular updates in a number of upper level laboratory courses at James Madison University.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.