The long term goals of this research include the development of methodology to permit the folding, flexibility and dynamics of protein structure to be interrogated, and the development and use of fluorescent probes in novel ways to enhance our understanding of protein structure, dynamics and function. During the five years of proposed research, the focus of efforts will be on study of the mechanism of modified DHFRs through steady state, pre-steady state and single molecule kinetic studies. Fluorescence spectroscopy will be used to study the protein conformational changes and their association with the key steps in DHFR catalysis.

Public Health Relevance

The present project is aimed at describing and quantifying the particular sequence, structural and dynamic properties that operate to enhance enzymatic catalysis. The proposed studies will provide important insights into enzyme function and will foster protein design and drug discovery efforts. The enzyme DHFR, which is the target of important antineoplastic and antimicrobial drugs, will be the focus of our efforts.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM092946-04
Application #
8450124
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Lewis, Catherine D
Project Start
2010-04-01
Project End
2014-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
4
Fiscal Year
2013
Total Cost
$300,974
Indirect Cost
$52,583
Name
Arizona State University-Tempe Campus
Department
Miscellaneous
Type
Organized Research Units
DUNS #
943360412
City
Tempe
State
AZ
Country
United States
Zip Code
85287
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Talukder, Poulami; Chen, Shengxi; Liu, C Tony et al. (2014) Tryptophan-based fluorophores for studying protein conformational changes. Bioorg Med Chem 22:5924-34
Talukder, Poulami; Chen, Shengxi; Arce, Pablo M et al. (2014) Efficient asymmetric synthesis of tryptophan analogues having useful photophysical properties. Org Lett 16:556-9
Liu, C Tony; Wang, Lin; Goodey, Nina M et al. (2013) Temporally overlapped but uncoupled motions in dihydrofolate reductase catalysis. Biochemistry 52:5332-4
Chen, Shengxi; Fahmi, Nour Eddine; Bhattacharya, Chandrabali et al. (2013) Fluorescent biphenyl derivatives of phenylalanine suitable for protein modification. Biochemistry 52:8580-9
Chen, Shengxi; Fahmi, Nour Eddine; Wang, Lin et al. (2013) Detection of dihydrofolate reductase conformational change by FRET using two fluorescent amino acids. J Am Chem Soc 135:12924-7
Stojkovic, Vanja; Perissinotti, Laura L; Willmer, Daniel et al. (2012) Effects of the donor-acceptor distance and dynamics on hydride tunneling in the dihydrofolate reductase catalyzed reaction. J Am Chem Soc 134:1738-45
Chen, Shengxi; Fahmi, Nour Eddine; Nangreave, Ryan C et al. (2012) Synthesis of pdCpAs and transfer RNAs activated with thiothreonine and derivatives. Bioorg Med Chem 20:2679-89
Hammes, Gordon G; Benkovic, Stephen J; Hammes-Schiffer, Sharon (2011) Flexibility, diversity, and cooperativity: pillars of enzyme catalysis. Biochemistry 50:10422-30
Stojkovic, Vanja; Perissinotti, Laura L; Lee, Jeeyeon et al. (2010) The effect of active-site isoleucine to alanine mutation on the DHFR catalyzed hydride-transfer. Chem Commun (Camb) 46:8974-6

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