The broad goal of this research is to provide new and detailed insights into the fundamentals of enzyme action by the approach of Raman spectroscopy. Technical innovations offer unprecedented opportunities for the development and application of the Raman method. These will be exploited here to investigate two main classes of enzymes, a dehalogenase that is of potential environmental import, since it degrades a chlorinated hydrocarbon; and flavoproteins, some of which are promising targets for drug design. In both areas, Raman difference spectroscopy will be used to obtain the Raman spectrum of a ligand bound to the enzyme in stable 1:1 complexes, or in reaction intermediates. Interpretation of the Raman data, aided by quantum mechanical calculations, will provide detailed information on the conformation, electron distribution and molecular interactions for the ligand. Preliminary studies reveal that several enzyme-ligand complexes exist in solution in more than one conformational state. The thermodynamic properties of these conformations will be defined by studying the variation of populations with temperature. Technical innovations also permit the construction of sensitive Raman microscopes that acquire the Raman spectrum of a sample using microscope optics. Preliminary experiments with the Raman microscope have shown that high-quality Raman spectra can be obtained from single protein crystals. Raman spectra of the flavoprotein parahydroxybenzoate hydroxylase were obtained from crystals in hanging drops in their growth chambers. The crystals were only 30 microns in dimensions. Both protein and flavin peaks could be seen in the crystal Raman spectra. Moreover, marker bands for the buried or solvent-exposed flavin group could be discerned in two different enzyme-ligand complexes, each known to favor the buried or exposed conformation. Thus, for both dehalogenase and flavoproteins we are proposing to use Raman data to compare the details of ligand chemistry for complexes in solution versus single crystals. We will also make a careful comparison of conformational populations, where more than one conformational state is found.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM054072-05
Application #
6128890
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Ikeda, Richard A
Project Start
1996-04-01
Project End
2004-03-31
Budget Start
2000-04-01
Budget End
2001-03-31
Support Year
5
Fiscal Year
2000
Total Cost
$299,040
Indirect Cost
Name
Case Western Reserve University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Heidari-Torkabadi, Hossein; Bethel, Christopher R; Ding, Zhe et al. (2015) ""Mind the Gap"": Raman Evidence for Rapid Inactivation of CTX-M-9 ?-Lactamase Using Mechanism-Based Inhibitors that Bridge the Active Site. J Am Chem Soc 137:12760-3
Heidari-Torkabadi, Hossein; Che, Tao; Lombardo, Michael N et al. (2015) Measuring propargyl-linked drug populations inside bacterial cells, and their interaction with a dihydrofolate reductase target, by Raman microscopy. Biochemistry 54:2719-26
Antonopoulos, Ioanna H; Warner, Brittany A; Carey, Paul R (2015) Concerted Protein and Nucleic Acid Conformational Changes Observed Prior to Nucleotide Incorporation in a Bacterial RNA Polymerase: Raman Crystallographic Evidence. Biochemistry 54:5297-305
Che, Tao; Rodkey, Elizabeth A; Bethel, Christopher R et al. (2015) Detecting a quasi-stable imine species on the reaction pathway of SHV-1 ?-lactamase and 6?-(hydroxymethyl)penicillanic acid sulfone. Biochemistry 54:734-43
Antonopoulos, Ioanna H; Murayama, Yuko; Warner, Brittany A et al. (2015) Time-resolved Raman and polyacrylamide gel electrophoresis observations of nucleotide incorporation and misincorporation in RNA within a bacterial RNA polymerase crystal. Biochemistry 54:652-65
Heidari Torkabadi, Hossein; Bethel, Christopher R; Papp-Wallace, Krisztina M et al. (2014) Following drug uptake and reactions inside Escherichia coli cells by Raman microspectroscopy. Biochemistry 53:4113-21
Sui, Xuewu; Kiser, Philip D; Che, Tao et al. (2014) Analysis of carotenoid isomerase activity in a prototypical carotenoid cleavage enzyme, apocarotenoid oxygenase (ACO). J Biol Chem 289:12286-99
Che, Tao; Bethel, Christopher R; Pusztai-Carey, Marianne et al. (2014) The different inhibition mechanisms of OXA-1 and OXA-24 ?-lactamases are determined by the stability of active site carboxylated lysine. J Biol Chem 289:6152-64
Heidari Torkabadi, Hossein; Che, Tao; Shou, Jingjing et al. (2013) Raman spectra of interchanging ýý-lactamase inhibitor intermediates on the millisecond time scale. J Am Chem Soc 135:2895-8
Rodkey, Elizabeth A; McLeod, David C; Bethel, Christopher R et al. (2013) ?-Lactamase inhibition by 7-alkylidenecephalosporin sulfones: allylic transposition and formation of an unprecedented stabilized acyl-enzyme. J Am Chem Soc 135:18358-69

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