The objective of this proposal is to obtain useful structural information on biological macromolecules and their complexes with ligands under conditions where X-ray diffraction methods cannot be carried out. We will study the conformation and dynamical changes of proteins and nucleic acids in both solution and crystalline states. We believe that such information would be of great help in understanding the relation between the conformation, dynamics and biological function of these macro-molecules. This in turn should lead to a better understanding of the molecular basis of biological function and aid in the design of more rational health care. Our primary experimental method will be classical and ultraviolet resonance RAMAN (UVRR) spectroscopy. We propose to obtain the UVRR spectra of thiointermediates that are formed during enzymatic catalysis involving thiol containing enzymes. Such intermediates absorb light at 240 nm and produce excellent UVRR spectra using the 239 nm exciting line from a frequency modified Nd-YAG laser. We propose to start with the enzyme, glyceraldehyde-3-phosphate dehydrogenase in order to answer some fundamental questions about the structure of the thio-intermediates formed during catalysis and their interaction with the cofactors NAD+ and NADH at the active site. We propose to apply this new technique to intermediates formed during catalysis by metal containing enzymes. A second new Raman technique that we have developed measures the Raman spectrum of an enzyme single crystal that is immersed in a bath of mother liquor containing the substrate. An enzyme-substrate intermediate builds up to a steady state in the crystal as substrate diffuses in and product diffuses out. We have demonstrate the feasibility of this technique with single crystals of carboxypeptidase. In each experiment we hope to find evidence for the formation of intermediates of unusual structures which will explain the catalytic activity of the enzyme in a more complete manner. We further propose to develop a new method for the quantitative determination of trace amounts of DNA in an unusual A or Z type conformation in the presence of a preponderance of B form DNA by combining photochemical crosslinking experiments with UVRR spectroscopy.
