Synthesizing Caged-NAD Molecules to Conduct Laue Time-Resolved Crystallography Measurements on HMGCoA Reductase

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Time-resolved x-ray crystallography can be used to collect x-ray diffraction data at femtosecond timescales as an enzymatic reaction progresses in a crystal. Caged compounds are used to trap inactive substrates within crystals that can be phototriggered and released using laser pulses at specific wavelengths before collecting x-ray diffraction data. Crystallizing enzyme cofactors as caged compounds is a technique employed to initialize enzymatic reactions in conjunction with x-ray diffraction data collection, thus giving us temporal control over the reaction.Our research is aimed at studying the reaction mechanism of the enzyme HMGCoA reductase. The enzyme can undergo a conformational change inside a crystal after binding to the reaction substrates HMGCoA and NAD, thereby making it a suitable enzyme for time-resolved studies. In order to obtain time-resolved information about the reaction, we are currently trying to synthesize caged NAD molecules that undergo photolysis at wavelengths greater than the absorbance wavelength of the different protein residues in order to trigger cleavage of the substrate from the caged compound without perturbing the protein. Using the NAD binding proteins, NAD Glycohydrolase and ADP-Ribosyl Cyclase, we hope to synthesize caged NAD molecules using different Nicotinamide derivatives. In this study, we outline properties of Nicotinamide derivatives (solubility, size, steric effects and photocage linker lability) that affect the formation of Caged NAD molecules in NAD Glycohydrolase and ADP-Ribosyl Cyclase. By showcasing, how rate of formation of different caged NAD molecules differs with respect to these properties, we hope to optimize the conditions for forming caged-NAD molecules that can function effectively inside a crystal.