The long-term goals of this project are to develop spectroscopic methods for probing electric fields in proteins and to apply these methods to obtain quantitative information on fields and their effects on function at the active sites of several enzymes and green fluorescent proteins (GFPs). Electrostatic interactions impact every aspect of the structure and function of proteins, nucleic acids and membranes. Variations in the magnitude and direction of electric fields can significantly affect the rates of elementary processes such as electron and proton transfer, where charge moves over a substantial distance. Similarly, 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 is essential for catalysis. The landscape of electric fields steers the binding of substrates, inhibitors and allosteric effectors to macromolecules and directly affects binding constants. On a larger scale, electrostatic interactions affect protein folding, macromolecular interactions and the assembly of subunits into larger structures. The magnitudes of the electric fields in proteins and the variations in these fields at different sites can be enormous. While these variations and their absolute magnitudes are well appreciated by theorists, who have developed a large body of analytical, computational, and graphical methods to evaluate electrostatic potentials, it has proven to be more difficult to obtain quantitative experimental information on either local variations in electric fields in proteins or the time-dependent changes in these fields coupled to functionally-relevant changes in charge distribution. The proposed research outlines a series of approaches and targets that can address these core issues. Aim 1 outlines experiments that probe time-averaged and time-dependent electric fields in several enzymes: human aldose reductase and human aldehyde reductase (Sub-Aim 1A), both important to the control of diabetes, and ketosteroid isomerase (Sub-Aim 1B). The proposed work is focused on rigorously comparing measured and calculated fields by using vibrational Stark spectroscopy, discriminating between structurally-similar active sites by understanding electrostatic fields, and incisively probing the mechanism of an enzyme by employing electric field detectors close to the site where catalysis occurs. Aim 2 outlines measurements that probe time-dependent excited state proton transfer and electrostatics using novel GFP constructs. In part, this work continues a long-standing and high impact effort to understand the photophysics and photochemistry of GFP variants. In addition, we propose to extend this effort by using split GFPs to probe the assembly of the 2-barrel structure and by introducing unnatural amino acids at functionally interesting sites throughout the protein. PUBLIC HEALTH RELEVANCE We are investigating several proteins that have direct relevance to human health. In particular, we propose new approaches for characterizing and potentially differentiating electrostatics at the active sites of human aldose reductase and human aldehyde reductase. The former is a primary cause of the complications of diabetes, but selective inhibition of one enzyme relative to the other has, thus far, proven elusive. New experiments are also proposed for GFP which is the most widely used fluorescent protein for cell-based imaging.