This is a request for continuing support of a research program aimed at understanding the role of protein dynamics and electrostatics in biological function. Myoglobin (Mb) was chosen because it is an extremely well characterized, stable protein for which the genes and excellent expression systems are available. The proposed research is divided into three sections: 1. The role of the proximal ligand in heme protein function: we recently developed a simple method for replacing the proximal histidine in Mb with a very wide range of small organic ligands including methyl-substituted imidazoles, pyridine, phenol, furan, thiophene, and ethanethiol. With this method it is possible to radically alter the properties of Mb. The consequences for heme protein function are being investigated by a broad range of spectroscopic and structural methods. 2. New ligand binding pathways in Mb based on a random library: a random library of all single amino acid mutants of Mb has been prepared and screened for changes in ligand binding kinetics. Very large changes were observed for a surprisingly large fraction of these mutants; including many residues which are remote from the best-studied ligand binding pathway. These new pathways will be studied in order to understand the molecular features which regulate access of gaseous ligands to the heme pocket. 3. Solvation dynamics in proteins: the time-dependent solvation of charge is an essential feature of reactions in biological systems and is often the subject of molecular dynamics simulations. Dielectric relaxation in proteins can be quantitated by monitoring the time evolution of the Stokes shift of a fluorescent probe. We have used this method to measure the timescale for solvation of a dye bound in the heme pocket of apoMb from 20 ps to 20 ns. Large contributions to the solvation were observed over this entire timescale range, and there is strong evidence suggesting both faster and slower timescale contributions. We have recently obtained a mode-locked Ti:sapphire laser permitting fluorescence lifetime measurements with approximately 50 fs time resolution. Using this system we propose to extend the time resolution of the dynamic Stokes shift measurement in the dye-apoMb system to this timescale. This will permit a direct comparison of experimental data with predictions from molecular dynamics simulations.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM027738-18
Application #
2459326
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1980-08-01
Project End
1999-07-31
Budget Start
1997-08-01
Budget End
1998-07-31
Support Year
18
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Stanford University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
800771545
City
Stanford
State
CA
Country
United States
Zip Code
94305
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
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
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
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
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

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