Vibrational Stark effect (VSE) spectroscopy is a technique that allows us to probe the local electrostatic field at specific locations in and around proteins. In these experiments, we use nitrile vibrational probes that are site-specifically placed in proteins by post-translational modification of cysteine residues. Shifts in vibrational energy of the nitrile probe are related linearly to changes in local electrostatic fields caused by changes in structure, binding, catalysis, and inhibition. This work focuses on the application of VSE spectroscopy as a measurement tool in three well-studied protein systems, p21Ras, Arf1, and Staphylococcal Nuclease. When analyzed along with a suite of other analytical characterization techniques and activity measurements, VSE also allows us to measure the role of electrostatics in catalysis (in the case of p21Ras), folding within the interior of proteins (in the case of Staphylococcal Nuclease), and protein-protein interactions (in the case of the Arf1 system) and in order to better understand these and related classes of proteins and their role in complex biological processes.<br/>In the case of p21Ras, we probed the electrostatic mechanism of intrinsic guanosine triphosphate (GTP) hydrolysis in the wild-type and oncogenic mutations of the protein. We showed that different mutations to position 61 of p21Ras resulted in different electrostatic fields at the GTP binding site depending on the identity of the residue. Additionally, we analyzed the relationship between the change in the electrostatic field and the change in intrinsic hydrolysis rate, which is related to cellular apoptosis.<br/>In Staphylococcal Nuclease, we related our measurements of the electrostatic field in the interior of the protein to previous measurements of pKa shifts of ionizable residues buried within the hydrophobic core that are crucial to structure and function.<br/>Arf1 experiments are ongoing, but the goal is to measure the changes in electrostatics when Arf1 docks to its partner protein and also when a small molecule natural product, Brefeldin A (BFA), inhibits the complex. Altogether, these are well-studied model systems that represent diverse classes of proteins in which structure and function are driven by electrostatic forces that can be understood and quantified with VSE spectroscopy to enhance understanding of electrostatic forces in biological processes.

Vibrational Stark effect spectroscopy as a tool for enhancing understanding of electrostatics in complex protein systems

255th National Meeting (2018)

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