Radiolytic Footprinting Methods for Structural Mass Spectrometry

描述（由申请人提供）：我们开发了基于羟基自由基修饰蛋白质的结构质谱的创新方法。这些结构质谱法最近得到了广泛的接受，并且已经成熟，可以进一步发展。在这个提议中，我们将：提高蛋白质足迹方法的灵敏度~1000倍；将对接方法与蛋白质足迹数据相结合，探索蛋白质复合物的结构，并开发使用足迹检测蛋白质中水动力学的方法。我们的初步数据表明，使用增加的x射线通量密度和更短的曝光时间，可以在基态和光激活状态下检测G蛋白偶联受体紫红质。在Aim 1中，我们将使用增加的x射线通量密度来进一步探索伴随GPCR激活的结构和溶剂动力学以及向下游信号蛋白介导信息的信号传导的结构机制。我们的指导假设是，高度保守的结构基序，包括结合水重组，以提供一个高度控制的信号通道。在目标2中，我们将进一步开发一种新的O18水标记-辐射分解技术，以检查结构水的位置和动态以及视紫红质和肌动蛋白在多种生物状态下的大块水的交换特性。在Aim 3中，我们将完善靶向质谱方法来检测蛋白质足迹实验中的低丰度修饰，以增加这些实验常规检测到的氨基酸侧链的数量。在目标4中，我们将开发将足迹数据纳入结构确定的对接计算方法。我们的假设是，足迹数据的使用将推动在具有可比能量的竞争对接解决方案中正确选择正确的结构。优化的算法方法将用于模拟肌凝蛋白或cofilin与肌动蛋白的复合物以及视紫红质和转导蛋白的复合物。

DESCRIPTION (provided by applicant): We have developed innovative methods of structural mass spectrometry based on hydroxyl radical modification of proteins. These structural mass spectrometry methods have recently gained widespread acceptance and are ripe for further development. In this proposal we will: increase the sensitivity of protein footprinting methods ~1000 fold; integrate docking approaches with protein footprinting data to probe the structure of protein complexes and develop methods to examine the dynamics of water in proteins using footprinting. Our preliminary data has shown feasibility for the examination of the G- protein coupled receptor rhodopsin in its ground and photo-activated states using increased x-ray flux density and shorter exposure times. Within Aim 1 we will use increased x-ray flux density to further explore the structural and solvent dynamics accompanying GPCR activation and the structural mechanism of signaling that mediates information to downstream signaling proteins. Our guiding hypothesis is that highly conserved structural motifs that include bound waters are reorganized to provide a highly controlled signaling channel. In Aim 2 we will further develop a novel O18 water labeling- radiolysis technique to examine the locations and dynamics of structural waters and the exchange properties of bulk water in multiple biological states of interest for rhodopsin and actin. In Aim 3 we will perfect targeted MS approaches to detect low abundance modifications in protein footprinting experiments to enhance the number of amino acids side chains routinely detected by these experiments. In Aim 4 we will develop computational methods of docking that incorporate footprinting data in structure determination. Our hypothesis is that use of footprinting data will drive correct selection of the correct structure among competing docking solutions that are of comparable energies. Optimized algorithmic approaches will be used to model complexes of myosin or cofilin with actin and complexes of rhodopsin and transducin. PUBLIC HEALTH RELEVANCE: G-protein coupled receptors are the targets of close to half of all drugs; a better understanding of their activation mechanisms would have a major impact on health and disease. Advanced technologies to examine the structure and dynamics of all proteins are needed in biology and medicine to better correlate structure and function.