The Role of Electrostatic Fields at the Protein-Protein Interface

生物学功能来自活细胞拥挤环境中多种蛋白质的相互作用。 在后基因组时代，增强对蛋白质之间的合作相互作用的理解对于探索生物过程的复杂性是必要的。 例如，信号蛋白Ras负责传播导致细胞分裂的化学信息。 为此，Ras在其生命周期中与多种蛋白质结合并相互作用，以在“开启”（信号）和“关闭”（沉默）状态之间切换。然而，驱动和稳定这些蛋白质-蛋白质相互作用并指导其最终功能的物理机制在很大程度上是未知的。这些相互作用是由蛋白质结构产生的弱但长程静电场的分布实现的。改变这些相互作用的蛋白质的结构或化学序列的细微变化可能是毁灭性的;例如，阻止其与适当伴侣结合的人类Ras蛋白质的突变使其永久处于“开启”状态，导致不受控制的细胞分裂和肿瘤生长。 对Ras蛋白质-蛋白质界面形成和功能的物理机制的深入研究将产生两个重要的结果：1）对Ras蛋白质的功能产生全新的认识;和2）关于静电场在蛋白质中的作用的一般知识-蛋白质相互作用，然后可以应用到各种各样的其他生物相关的多蛋白质复合物。该研究项目的先进的多学科性质将使在实验和理论化学和生物学的接口出现的重要问题的探索。在实现这些目标的过程中，学生和博士后研究人员将接受多学科工具和技术的培训，这些工具和技术将成为他们自己科学生涯的基础。PI的实验室利用光谱技术研究产生静电场的分子水平机制，从而确定蛋白质-蛋白质界面的形成和特异性。 该研究小组使用这种技术来研究正常Ras蛋白与其在化学信号通路中的结合伴侣（所谓的“效应”蛋白）之间的相互作用，作为所有生物学上重要的蛋白质-蛋白质界面的模型系统。 在本项目中，PI将使用该技术研究已知致癌Ras突变体与其相应效应蛋白的异常界面的形成，以了解正常和病理性Ras突变体之间的差异。 了解的详细机制，负责Ras和其他蛋白质之间的接口的形成，特别是Ras的癌性突变如何改变这些接口的功能，将提供一个全新的视角静电场的作用，在复杂的，多蛋白组装的结构，功能和动力学。如何在蛋白质-蛋白质界面的静电场可能会改变通过选择性结合的小分子，该接口也将被调查。 这将通过关注天然产物布雷菲德菌素A与Ras类似物及其下游效应物的界面的结合和抑制来实现。实验数据将用于验证和改进预测蛋白质静电场的计算技术。

Biological function emerges from the interaction of multiple proteins in the crowded environment of a living cell. In the post-genomic era, enhanced understanding of the cooperative interaction between proteins is necessary to explore the complexity of biological processes. For example, the signaling protein Ras is responsible for propagating a chemical message that leads to, among other things, cell division. To do this, Ras binds to and interacts with multiple proteins in its lifecycle to switch between "on" (signaling) and "off" (silent) states. However, the physical mechanisms that drive and stabilize these protein-protein interactions and direct their resulting function are largely unknown. These interactions are enabled by the distribution of weak, but long-range electrostatic fields that are generated by the proteins' structures. Subtle changes in the structure or chemical sequence of a protein that alter these interactions can be devastating; for example, mutations to the human Ras protein that prevent its binding to the appropriate partner leave it permanently in the "on" state, causing uncontrolled cell division and tumor growth. It is believed that a fundamental investigation into the physical mechanisms of the formation and function of Ras-based protein-protein interfaces will have two important outcomes: 1) generation of an entirely new understanding of the function of this specific protein ; and 2) general knowledge about the role of electrostatic fields in protein-protein interactions that can then be applied to a wide variety of other biologically relevant multiprotein complexes. The advanced multidisciplinary nature of this research project will enable the exploration of important questions that arise at the interface of experimental and theoretical chemistry and biology. In the process of achieving these goals, students and postdoctoral researchers will be trained in multidisciplinary tools and techniques that will form the foundations of their own scientific careers.The PI's laboratory utilizes spectroscopic techniques to study the molecular-level mechanisms that generate the electrostatic fields, which in turn determine the formation and specificity of protein-protein interfaces. The research group has used this technique to study the interactions between normal Ras proteins and their binding partners in the chemical signaling pathway (so called "effector" proteins) as a model system for all biologically important protein-protein interfaces. In this project, the PI will use this technique to investigate the formation of abnormal interfaces of known cancer-causing mutants of Ras with their appropriate effector proteins in order to understand the differences between normal and pathological Ras mutants. Understanding the detailed mechanisms that are responsible for the formation of an interface between Ras and other proteins and, in particular, how cancerous mutations of Ras alter the function of these interfaces, will provide an entirely new perspective on the role of electrostatic fields in the structure, function, and dynamics of complex, multiprotein assemblies. How electrostatic fields at the protein-protein interface may be altered through the selective binding of small molecules to that interface will also be investigated. This will be accomplished by focusing on the binding and inhibition of the natural product brefeldin A to the interface of a Ras analog with its downstream effector. The experimental data will be used to validate and refine computational techniques for predicting protein electrostatic fields.