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.

生物功能来自活细胞拥挤的环境中多种蛋白质的相互作用。 在基因组后时代，对蛋白质之间的合作相互作用的增强是为了探索生物过程的复杂性。 例如，信号蛋白RAS负责传播一条化学消息，该化学消息除其他外部外分裂。 为此，RAS与其生命周期中的多个蛋白质结合并与多个蛋白质相互作用，以在“ ON”（信号）和“ OFF”（静音）状态之间切换。但是，驱动和稳定这些蛋白质 - 蛋白质相互作用并指导其产生功能的物理机制在很大程度上未知。这些相互作用是通过蛋白质结构产生的弱但远程静电场的分布来实现的。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，防止其与适当伴侣结合的人类RAS蛋白的突变使其永久性地处于“ ON”状态，从而导致不受控制的细胞分裂和肿瘤生长。 据信，对基于RAS的蛋白质 - 蛋白质界面的形成和功能的物理机制进行了基本研究将有两个重要的结果：1）对该特定蛋白质的功能产生全新的理解； 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与生物学相关的多蛋白复合物。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界面上出现的重要问题探索。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，这些工具和技术将构成其自身科学职业的基础。PI的实验室利用光谱技术来研究分子水平的机制，这些机制会产生静电场，从而确定形成和特定的蛋白质练习，从而确定静电性的效率。 研究小组已使用该技术研究化学信号途径中正常RAS蛋白与其结合伴侣之间的相互作用（所谓的“效应子”蛋白）作为所有生物学上重要的蛋白质蛋白接口的模型系统。 在该项目中，PI将使用该技术来研究已知的癌症引起癌症突变体的异常界面及其适当的效应蛋白，以了解正常和病理RAS突变体之间的差异。 了解负责RAS与其他蛋白质之间界面形成的详细机制，尤其是RAS的癌性突变如何改变这些接口的功能，将为静电场在结构，功能和复杂动力学的结构，功能和动力学中的作用提供全新的观点。还将研究如何通过小分子与该界面的选择性结合来改变蛋白质蛋白界面的静电场。 这将通过重点关注天然产物Brefeldin A与其下游效应子的界面的结合和抑制来实现。实验数据将用于验证和完善计算技术，以预测蛋白质静电场。

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.