Vibrational Stark Effects for Diverse Carbonyl Probes Applied to the Re-Interpretation of IR and Raman Data in Terms of Electric Fields at Enzyme Active Sites

Abstract

Carbonyl groups are ubiquitious in biology and found in many different forms including ketones, oxoesters (e.g. lipids, acyl-intermediates), thioesters (e.g. acetyl-CoA, fatty acid biosynthesis), and amides (e.g. DNA, RNA, proteins). Previous work in our lab has utilized the vibrational Stark effect (VSE) of ketones on non-natural amino acids [1] and substrate analogs [2] to quantify the electric field within several proteins, based on the vibration's linear Stark tuning rate. In order to further probe diverse biophysical systems and environments, we have measured the sensitivity of a functionally-relevant collection of carbonyl-containing compounds by vibrational Stark spectroscopy (VSS) to obtain the Stark tuning rate, and further calibrated them to an absolute electric field utilizing vibrational solvatochromism and MD simulations. The VSE provides a quantitative framework for interpreting observed frequency shifts in biophysical systems, providing physical insights into binding and catalysis. As such, this calibration to an electric field provides a unifying model with which to re-examine a substantial body of literature on highly unusual IR and Raman data of inhibitors at the enzyme active site, which have been previously attributed to substrate polarization and bond distortion [e.g. 3]. The generalizability of this interpretation to many enzymatic systems further suggests the importance of enzyme electrostatics in modulating function and may have significant applications in protein design and engineering.