RAMAN STUDIES OF ENZYME COMPLEXES IN SOLUTION & CRYSTALS

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DESCRIPTION: (Verbatim from the Applicant's Abstract)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.