The Role of Electrostatic Fields at the Protein-Protein Interface

静电场在蛋白质-蛋白质界面的作用

• 批准号: 1714555
• 负责人: Lauren Webb
• 金额: $ 61.45万
• 依托单位: University of Texas at Austin
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1714555&HistoricalAwards=false
• 关键词: Role; Electrostatic; Fields; Protein; Interface

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.

生物功能来自于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，加强对蛋白质之间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负责传播一种化学信息，导致细胞分裂等。为了做到这一点，RAS在其生命周期中与多个蛋白质结合并相互作用，在“开”(信号)和“关”(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结果功能的物理机制在很大程度上是未知的。这些相互作用是通过蛋白质结构产生的弱而远距离的静电场的分布来实现的。蛋白质结构或化学序列的细微变化可能会改变这些相互作用，这可能是毁灭性的；例如，人类RAS蛋白的突变阻止了它与适当的伴侣结合，使其永久处于“开启”状态，导致细胞分裂和肿瘤生长失控。人们认为，对基于RAS的蛋白质-蛋白质界面形成和功能的物理机制的基础研究将有两个重要的结果：1)对这种特定蛋白质的功能有一个全新的理解；2)关于静电场在蛋白质-蛋白质相互作用中的作用的一般知识，然后可以应用于各种其他生物相关的多蛋白质复合体。这一研究项目的先进的多学科性质将使人们能够探索在实验和理论化学和生物学的交界处出现的重要问题。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，这些工具和技术将为他们自己的科学生涯奠定基础。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，这反过来又确定蛋白质-蛋白质界面的形成和特异性。该研究小组使用这项技术来研究正常RAS蛋白与其在化学信号通路中的结合伙伴(即所谓的效应蛋白)之间的相互作用，作为所有具有生物重要性的蛋白质-蛋白质界面的模型系统。在这个项目中，PI将使用这项技术来研究已知的RAS致癌突变与其适当的效应蛋白之间的异常界面的形成，以了解正常和病理RAS突变之间的差异。了解RAS和其他蛋白质之间形成界面的详细机制，特别是RAS的癌变如何改变这些界面的功能，将为理解静电场在复杂的多蛋白质组装的结构、功能和动力学中的作用提供一个全新的视角。还将研究如何通过小分子与蛋白质界面的选择性结合来改变蛋白质-蛋白质界面上的静电场。这将通过关注天然产物布雷菲尔丁A与RAS类似物及其下游效应器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。

项目成果

Quantitative Measurement of Intrinsic GTP Hydrolysis for Carcinogenic Glutamine 61 Mutants in H-Ras

• DOI: 10.1021/acs.biochem.8b00878
• 发表时间: 2018-11-06
• 期刊: BIOCHEMISTRY
• 影响因子: 2.9
• 作者: Novelli, Elisa T.;First, Jeremy T.;Webb, Lauren J.
• 通讯作者: Webb, Lauren J.

Investigation of GTP-dependent dimerization of G12X K-Ras variants using ultraviolet photodissociation mass spectrometry

• DOI: 10.1039/c9sc01032g
• 发表时间: 2019-09-14
• 期刊: CHEMICAL SCIENCE
• 影响因子: 8.4
• 作者: Mehaffey, M. Rachel;Schardon, Christopher L.;Brodbelt, Jennifer S.
• 通讯作者: Brodbelt, Jennifer S.

Structural Evaluation of Protein/Metal Complexes via Native Electrospray Ultraviolet Photodissociation Mass Spectrometry

• DOI: 10.1021/jasms.0c00066
• 发表时间: 2020-05-06
• 期刊: JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
• 影响因子: 3.2
• 作者: Crittenden, Christopher M.;Novelli, Elisa T.;Brodbelt, Jennifer S.
• 通讯作者: Brodbelt, Jennifer S.