Proceedings of nanoGe September Meeting 2015 (NFM15)
Publication date: 8th June 2015
Electrostatic interactions impact every aspect of the structure and function of proteins, nucleic acids, and membranes. The transition states for many enzyme-catalyzed reactions involve a change in the distribution of charge relative to the starting material and/or products, and the selective stabilization of charge-separated transition states may be essential for catalysis. The magnitudes of the electric fields in proteins and the variations in these fields at different sites are predicted to be enormous, but it is a challenge to obtain quantitative experimental information on these fields. 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 [1]. The strategy involves deploying site-specific vibrational probes 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 interactions such as O-H··p interactions that stabilize protein structures. By measuring vibrational solvatochromism in conjunction with simulations, we obtain information on absolute fields and this can be applied to obtain information on functionally relevant electric fields at the active site of enzymes. Using ketosteroid isomerase (KSI) as a model system, we correlate the field sensed at the bond involved in enzymatic catalysis with the rate of the reaction it catalyzes, including variations in this rate in a series of mutants [2]. This provides the first direct connection between electric fields and function: for this system electrostatic interactions are a dominant contribution to catalytic proficiency. Applications to other systems will be discussed.
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