Measuring electrostatics in complex protein systems using vibrational Stark effect spectroscopy
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The non-covalent interactions between and within proteins are important because of their specificity and their ability to control protein structure and function. The measurement of electric fields, which describe these complex interactions, is crucial to understanding the physical properties of proteins such as folding, catalysis, and multimolecular interactions. Our goal is to understand and ultimately exploit these complex interactions for therapeutic value. In particular, our group has been interested in small GTPase signaling proteins, such as Ras, because of their known oncogenic properties, complex network of binding partner proteins, and their difficulty as a drug target. In particular the highly specific protein-protein interactions inherent in the signaling role of Ras and other GTPase proteins are interesting potential drug targets that could avoid the toxicity of competitive inhibition at the GTP binding site. To this end, we have made use of vibrational Stark effect spectroscopy, a technique that directly reports on electrostatic environment, to measure the electric fields in Ras and other GTPases by site-specifically incorporating small nitrile vibrational probes into proteins. We have related the quantitative field measurements to protein activity to elucidate the role of electric fields in the intrinsic hydrolysis mechanism of oncogenic Ras mutants. We also designed experiments to investigate the role of electrostatics in driving specific protein-protein interactions and their inhibition by the small molecule Brefeldin A in the GTPase protein Arf. Finally, we developed the Staphylococcus aureus protein staphylococcal nuclease as a robust model system for experiments aimed at better understanding how pK [subscript a] and nitrile probes respond to their local electrostatic environment. Together, this work demonstrates the importance of electrostatic forces in protein function and highlights how vibrational Stark effect spectroscopy can be applied effectively to interesting and relevant protein systems to observe and exploit the physical properties of proteins for therapeutic benefit.