The Role of Electrostatic Fields at the Protein-Protein Interface

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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.