This research program aims to obtain quantitative information of electrostatic interactions and dynamics in proteins. Significant new opportunities result for the availability of a cDNA clone for human myoglobin (mb) and an efficient system for producing the protein in E. coli. Part of the proposed research concerns structural and functional characterization of this important human protein. A fundamental question is the mechanism by which diatomic ligands enter and exit the iron binding site. Since there are no channels in the static X-ray structure, fluctuations are required for ligand access. This is a case where protein dynamics and biological function are directly related. Specific proposals of protein residues whose dynamics are crucial for ligand access are being tested systematically by site-specific mutagenesis. Ligand binding dynamics are measured from the femptosecond to kilisecond timescales by a wide range of spectroscopic techniques. Inherent in studies using site- specific mutagenesis is a bias that certain residues are more important than others. In order to avoid this bias a random mutagenesis strategy is outlined along with an approach to mass screening of mutants on the basis of ligand recombination kinetics. A second major area of research is to obtain quantitative information of electrostatic interactions in proteins using Mb mutants as a working model. Buried, potentially charged or polar amino acids are inserted in the sequence at defined locations. Information on the polarity of the protein interior and its effect on inter-residue electrostatic interactions is obtained by quantitative analysis of spectral and pKa shifts and changes in redox thermodynamics. A novel combination of non- photochemical holeburning and Stark effect spectroscopy is outlined to obtain information on the distribution of electrostatic fields in the heme pocket and the contribution of individual amino acid residues to the total electrostatic field. An approach to obtaining information of the time-dependent response of the protein interior to a sudden change in polarity is outlined. This is an important aspect of understanding the role played by the protein dielectric in regulating reactions such as electron transfer. An extension of earlier work in Mb and in homogeneous solution is proposed which involves measurement of the time-dependence of the emission spectrum of probe molecules in the heme pocket. Advances in ultra-fast fluorescence methods, theories of liquid dynamics and the possibility to examine the role of individual amino acid residues by site-specific mutagenesis make this possible.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Biophysics and Biophysical Chemistry A Study Section (BBCA)
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Stanford University
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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

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