Nucleoplasmin (Np) is a pdtameric molecule (molecular weight 110 kDa) that in solution forms decamers and is the most abundant protein in Xenopus oocytes. This protein is also the founding member of a small family of chaperones, which function in the assembly of nucleosomes and storage of histone octamers. The mechanism and details of these interactions are unknown. In order to address these issues, we have over-expressed Np and made a truncated core by chymotrypsinolysis. This core molecule is predominantly a pentamer in solution, but when crystallized in C2 it forms a decamer with non-crystallographic 522 point group symmetry. After quite a bit of work, we concluded that this crystal form had both native and derivative-based non-isomorphism problems, and that a single crystal-based method would be needed to solve the structure. Hence, we made seleno-methionine substituted Np-cores (3 Met/core monomer). This protein waschecked by electrospray mass spectrometry at BUSM, which indicated an 80% occupancy after purification. The data further suggested that there were 3 sites, with I or 2 sites being more resistant to oxidative loss of the seleruium. Crystals were grown and MAD data sets were collected at beamline X12C (Brookhaven NSLS), where X-ray fluorescence confirmed the selenium substitution. These data have led to the first 3 angstrom map of the pentamer/decamer, after 5-fold averaging. This work is still in progress, but when finished should reveal insights into why Np is unusually thermo- and chemically stable, how the pentamer and decamer are formed and, perhaps, a novel fold for this small chaperone family. This project represents the first step in a long-term project in which the structure of large complexes containing Np, histone octamers and the NI co-chaperone will be solved using a combination of electron cryo-microscopy and X-ray crystallography. This approach should reveal how these chaperone complexes function to stabilize the histone octamer under conditions where it would normally be disassembled, thereby preparing the octamers for incorporation into nucleosomes, the fundamental budding block of chromatin.
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