W0256

Purification, Crystallization, and Preliminary X-Ray Analysis of Intact Native Canavalin. Marc Pusey¹, Jennifer Dowell², Jose A. Gavira², Joseph Ng², ¹Biophysics SD46, NASA/MSFC, Huntsville, AL 35812, ²Laboratory of Structural Biology, Univ. of Alabama in Huntsville, Huntsville, AL 35899.

The protein canavalin is a 7S vicilin, from the Jack Bean, Canavalis ensiformis. Canavalin is described as a seed storage protein, an energy source for a developing seed, as no other known activity or function has been found. The protein was first isolated and crystallized by Sumner and Howell (J. Biol. Chem. 113, 607-610, 1936). Canavalin spontaneously crystallizes after proteolytic cleavage at neutral pH, which removes residues 1-46, 224-245, and 325-330 and produces peptides of ~25, 13, and 12 kDa. Preliminary gel filtration experiments indicated the presence of nucleic acid with the uncleaved protein. We developed a dual column procedure, ion exchange followed by hydroxy apatite chromatography, that effectively removes the nucleic acid and yields an essentially pure uncut canavalin preparation with an OD 280/260 ratio of ~1.9-2.0. Standard crystallization screens using this material gave a number of positive results having a common requirement for alcohols and Mg2+ on, with crystals typically appearing within a day or less. Optimization experiments to date have shown that we can obtain crystals from pH 6.5 to pH 8.2, using MPD from 5 to 20% and 0.05 to 0.2M Mg2+ (sulfate or acetate). The crystals are of space group P212121, unit cell dimensions, and a complete data set to 1.7 Å resolution has been collected. Most importantly, the crystals are not twinned, a persistent problem with the most commonly obtained rhombohedral form of proteolytically cleaved canavalin.