The Vibrational Stark Effect in Carboxylate Anions: A Sensitive Electrostatic Probe for Protein Environments

The electrostatic environment in proteins is critical for understanding ligand binding and enzyme catalysis and is required to inform the design of enzyme inhibitors with a desired mechanism of action. The vibrational Stark effect (VSE) has been utilized for extracting insights into the electrostatics in protein environments by including functional groups with detectable vibrational frequency responses to nearby electric fields. Various classical VSE probes have yielded much insight into enzymes such as beta-lactamase. However, among the most-used probes, nitriles have a stronger frequency shift than expected when accepting hydrogen bonds, and carbonyls are IR active in a frequency region that is generally cluttered in proteins.



In biological conditions with a pH near 7, carboxylic acids will deprotonate into carboxylate anions, which have an outsized electrostatic function as side chains of proteins and the binding of various druglike compounds in both soluble and membrane proteins. Carboxylates are not only biologically ubiquitous, but also have an intense IR absorbance at the frequencies of the asymmetric and symmetric COO- stretches, making them excellent candidates for VSE probes. In this work, we have performed theoretical and experimental studies of the vibrational Stark properties of carboxylate anions. Both vibrational Stark spectroscopy and solvatochromism measurements show that the asymmetric stretch of carboxylate anions functions as a robust VSE probe. We demonstrate that carboxylate probes are suitable tools in anionic-binding regions where other classical probes may not reach, such as the one found in the human aldose reductase enzyme (hALR2).