Growth of Large, Perfect Protein Crystals for Neutron Crystallography

Superoxide dismutases (SODs) are important antioxidant enzymes that protect all living cells against toxic oxygen metabolites, also known as reactive oxygen species (ROS). SODs are the first defense against propagation of damaging oxidative reactions through elimination of superoxide. Superoxide is generated through normal metabolism and/or ionizing radiation. Each catalytic cycle dismutes two molecules of superoxide to oxygen and hydrogen peroxide via cyclic reduction and oxidation half reactions using the active site metal ion. Humans have Cu/ZnSOD in the cytosol and extracellular spaces and MnSOD in their mitochondria. Mutations in SOD lead to aging and degenerative diseases such as amyotrophic lateral sclerosis, diabetes, and cancer. This flight proposal will provide the critical crystal samples needed for a detailed study of human SOD. Despite the biological and medical importance of SOD, the complete enzymatic mechanism is still unknown. Precise structural data are needed. The binding sites of the diatomic substrate and product as well as the source of the protons in the reaction have been studied, but their exact identification has not been possible. This detailed information can only be determined by neutron diffraction. Complexes of human MnSOD including structural intermediates and mutants will be the targets for large volume crystal (≥ 1mm3) growth for structure determination by neutron macromolecular crystallography (NMC). The quiescent environment afforded by microgravity is known to grow crystals large enough for neutron studies; not only are they large but their quality approaches perfection. In 2001, the Borgstahl laboratory successfully grew large crystals of SOD using microgravity conditions on the International Space Station (ISS). With NASA’s renewed interest in implementing the microgravity environment on the ISS for protein crystal growth we would like to move forward with these exciting early microgravity crystallization results for SOD. Existing crystallization hardware that uses the Granada Crystallization Boxes (GCB) for capillary counterdiffusion protocols will be used to achieve these goals. A microgravity environment is essential to form a stable supersaturation gradient to obtain the large, high quality crystals required for NMC. Then NMC will be performed with collaborators at Oak Ridge National Laboratory (ORNL). The principal outcome will be to identify the role of hydrogen atoms in enzymatic activity, discern superoxide from peroxide, and water from hydroxide ion by their protonation state and decipher a structure-based mechanism for human MnSOD more precisely than from previous X-ray crystallographic models determined from Earth-grown crystals. These contributions will also provide criteria needed for the protein engineering of desirable properties into enzymatic metal centers for proton coupled electron transfer.

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