Structure and stability of protein complexes in solution and the gas phase

大多数生物过程，包括免疫反应、细胞间通讯、炎症以及细菌和病毒感染，都涉及生物分子之间的结合，形成特定的非共价复合体。这些络合物的结构和稳定性是由结合伙伴之间的多种作用力(如氢键、离子和范德华相互作用)的协同作用以及与结合伙伴的溶剂壳层相关的溶剂分子的置换和重组决定的。对这些力量及其导致的结构的了解，对于全面理解生物过程是至关重要的。我们实验室的研究重点是开发和应用基于质谱学(MS)的技术，在体外检测特定的非共价蛋白质复合体(如蛋白质配体和多蛋白质复合体)，并表征它们在溶液和气相中的结构和稳定性。这项建议描述了基于电喷雾电离(ES)-MS技术的新的实验方法的发展，以量化溶液中非共价蛋白质相互作用的动力学和热力学参数。具体地说，我们将开发一种直接和灵敏的ES-MS方法来量化蛋白质与大分子(例如蛋白质和其他生物聚合物)相互作用的热力学参数。我们还将开发一种新的方法，基于温度跳跃松弛方法和ES-MS，来量化非共价蛋白质相互作用的动力学参数。从这些方法和其他方法中获得的动力学和热力学数据将使我们能够更详细地了解溶液中蛋白质识别所依据的结构-功能关系。我们还将使用红外多光子解离(IRMPD)光谱来探索去溶解的非共价蛋白质复合体的结构。特别是，我们将评估这项技术探索气态蛋白质复合体中分子间相互作用的性质的能力。通过比较气相溶液中识别的相互作用，将获得对溶剂在生物识别中的作用的新的见解。

Most biological processes, including the immune response, cell-cell communication, inflammation and bacterial and viral infections, involve the association of biomolecules to form specific, non-covalent complexes. The structure and stability of these complexes are determined by the concerted action of many forces (e.g. hydrogen bonds, ionic and van der Waals interactions) between binding partners and from the displacement and reorganization of solvent molecules associated with the solvent shell of the binding partners. An understanding of these forces and the structures they lead to is essential to a complete understanding of biological processes. Research in our laboratory focuses on the development and application of mass spectrometry (MS)-based techniques to detect specific, non-covalent protein complexes (e.g. protein-ligand and multi-protein complexes) in vitro and to characterize their structure and stability in solution and the gas phase. This proposal describes the development of novel experimental methodologies, based on the electrospray ionization (ES)-MS technique, to quantify the kinetic and thermodynamic parameters for non-covalent protein interactions in solution. Specifically, we will develop a direct and sensitive ES-MS approach to quantify the thermodynamic parameters for protein interactions with macromolecules (e.g. proteins and other biopolymers). We will also develop a new method, based on the temperature-jump relaxation approach and ES-MS, to quantify the kinetic parameters for non-covalent protein interactions. The kinetic and thermodynamic data available from these and other methods will allow us to develop a more detailed picture of the structure-function relationships that underlie protein recognition in solution. We will also explore the structure of desolvated non-covalent protein complexes using infrared multiphoton dissociation (IRMPD) spectroscopy. In particular, we will assess the ability of this technique to probe the nature of intermolecular interactions within the gaseous protein complexes. From a comparison of the interactions identified in solution in the gas phase, new insights into the role of solvent in biological recognition will be gained.