Vibrational Stark Spectroscopy Directly Probes Electric Fields in Proteins

Abstract

We have developed vibrational Stark effect (VSE) spectroscopy to probe electrostatics and dynamics in organized systems, in particular in proteins where they can report on functionally important electric fields. The strategy involves deploying site-specific vibrational probes (-C≡N, -C-D, -C=O and -C-F) whose sensitivity to an electric field is measured in a calibrated external electric field by VSE spectroscopy. This gives the magnitude of the vibrational frequency shift associated with an electric field change in a protein, e.g. by making a mutation, changing pH, ligand binding, etc., projected along the bond axis, which is typically determined by x-ray crystallography. This concept can also be used to estimate the electrostatic contribution to non-covalent X-H••π interactions that stabilize protein structures. Recent results in which we attempt to calibrate the absolute magnitude of the field will be presented, along with complications that arise from non-electrostatic contributions to the frequency shifts. This requires a substantial development of simulations methods in parallel with experiments. By fully understanding the origins of these effects, they can be applied to obtain information on functionally-relevant fields at the active site of enzymes (see also presentation by Fried, Bagchi and SGB). This approach provides experimental benchmarks for high-level simulations which have suggested a critical role for electric fields and electric field gradients in biological function.