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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM027738-33A1
Application #
8502147
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Smith, Ward
Project Start
1980-08-01
Project End
2017-01-31
Budget Start
2013-04-01
Budget End
2014-01-31
Support Year
33
Fiscal Year
2013
Total Cost
$365,482
Indirect Cost
$117,482
Name
Stanford University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
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
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
Fried, Stephen D; Bagchi, Sayan; Boxer, Steven G (2014) Extreme electric fields power catalysis in the active site of ketosteroid isomerase. Science 346:1510-4
Levinson, Nicholas M; Boxer, Steven G (2014) A conserved water-mediated hydrogen bond network defines bosutinib's kinase selectivity. Nat Chem Biol 10:127-32
Oltrogge, Luke M; Wang, Quan; Boxer, Steven G (2014) Ground-state proton transfer kinetics in green fluorescent protein. Biochemistry 53:5947-57
Wang, Lu; Fried, Stephen D; Boxer, Steven G et al. (2014) Quantum delocalization of protons in the hydrogen-bond network of an enzyme active site. Proc Natl Acad Sci U S A 111:18454-9
Sigala, Paul A; Fafarman, Aaron T; Schwans, Jason P et al. (2013) Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site. Proc Natl Acad Sci U S A 110:E2552-61

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