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 seveal 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 contours of electric fields steer the binding of substrates, inhibitors and allosteric effectors to macromolecules and directly affect 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 thee fields at different sites are predicted to be enormous, but it is a challenge to obtain quantitativ 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 development of methodology for introducing and characterizing vibrational probes for electric fields in proteins. These methods are used to probe fields in several enzymes. The proposed work focuses on a rigorous comparison between measured and calculated fields and incisive studies of the role of electrostatic interactions in catalytic mechanisms.
Aim 2 outlines strategies to understand the mechanism(s) of the recently discovered coupling between light-driven structural dynamics of peptide-protein re-assembly or dissociation in split GFP. A wide range of structural and spectroscopic techniques will be deployed to elucidate the mechanism(s) of this coupling. The split semi-synthetic GFP system will also be used to probe the assembly of the ?-barrel itself using the built-in reporter chromophore, the origin(s) of color tuning, and both ground and excited state proton transfer. These are achieved by introducing unnatural amino acids as probes or perturbations at functionally interesting sites throughout the protein, now enabled by these semi-synthetic systems. Applications of these novel systems for advanced imaging and as modulators of enzyme function that affect cell physiology with light are described.

Public Health Relevance

We are developing methods that probe the electrostatic field at the active site of enzymes, often using drugs that display the probes that sense the field. This is a new quantitative approach for discriminating active sites in targets of direct biomedical relevance, such as human aldose reductase (diabetes) and tyrosine kinases (cancer). Experiments are also proposed for green fluorescent protein (GFP), which is the most widely used fluorescent protein for cell-based imaging and where systems we have created can be used to modulate cell physiology.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Smith, Ward
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Stanford University
Schools of Arts and Sciences
United States
Zip Code
Deng, Alan; Boxer, Steven G (2018) Structural Insight into the Photochemistry of Split Green Fluorescent Proteins: A Unique Role for a His-Tag. J Am Chem Soc 140:375-381
Schneider, Samuel H; Kratochvil, Huong T; Zanni, Martin T et al. (2017) Solvent-Independent Anharmonicity for Carbonyl Oscillators. J Phys Chem B 121:2331-2338
Fried, Stephen D; Boxer, Steven G (2017) Electric Fields and Enzyme Catalysis. Annu Rev Biochem 86:387-415
Lin, Chi-Yun; Both, Johan; Do, Keunbong et al. (2017) Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs). Proc Natl Acad Sci U S A 114:E2146-E2155
Schneider, Samuel H; Boxer, Steven G (2016) Vibrational Stark Effects of Carbonyl Probes Applied to Reinterpret IR and Raman Data for Enzyme Inhibitors in Terms of Electric Fields at the Active Site. J Phys Chem B 120:9672-84
Wu, Yufan; Boxer, Steven G (2016) A Critical Test of the Electrostatic Contribution to Catalysis with Noncanonical Amino Acids in Ketosteroid Isomerase. J Am Chem Soc 138:11890-5
Fried, Stephen D; Boxer, Steven G (2015) Measuring electric fields and noncovalent interactions using the vibrational stark effect. Acc Chem Res 48:998-1006
Fried, Stephen D; Boxer, Steven G (2015) BIOPHYSICS. Response to Comments on ""Extreme electric fields power catalysis in the active site of ketosteroid isomerase"". Science 349:936
Wu, Yufan; Fried, Stephen D; Boxer, Steven G (2015) Dissecting Proton Delocalization in an Enzyme's Hydrogen Bond Network with Unnatural Amino Acids. Biochemistry 54:7110-9
Oltrogge, Luke M; Boxer, Steven G (2015) Short Hydrogen Bonds and Proton Delocalization in Green Fluorescent Protein (GFP). ACS Cent Sci 1:148-56

Showing the most recent 10 out of 86 publications