The Crystal Structures of Thaumatin with the Stereoisomers of Tartrate

Charles Ginsberg, Yeshiva University Neer Asherie, Yeshiva University Jean Jakoncic, Brookhaven National Lab Aryeh Greenbaum, Yeshiva University Vivian Stojanoff, Brookhaven National Lab Sam Blass, Yeshiva University

Abstract Thaumatin is a small, globular protein (22 kDa) that is used as a model system to study protein crystallization because it crystallizes rapidly in the presence of tartrate ions. Our recent experimental results show that the solubility and habit of crystalline thaumatin are strongly affected by which tartrate stereoisomer is used as a precipitant. To elucidate these results, we have purified thaumatin and crystallized it by both batch and microbatch methods with different precipitants: L-, D-, and meso-tartrate, DL-tartrate (racemate) and no tartrate. Batch experiments suggested that high quality crystals could form in 10 mM sodium phosphate (pH=7.3). Further optimization was carried out in microbatch experiments, where we changed solution conditions such as temperature and the concentration of both precipitant and additive (glycerol). Crystal nucleation, growth and habits were monitored by brightfield and polarized light microscopy. When L-tartrate was used as a precipitant, bipyramidal crystals formed rapidly in agreement with previous studies. With D-tartrate (for which there are no published results) prismatic crystals formed, and these eventually grew to a more stubby shape. Over time, bipyramidal crystals also appeared. Racemic mixtures of DL-tartrate produced bipyramids and prisms whose proportions depended on the solution conditions. Meso-tartrate produced prisms, while in the absence of any tartrate ions thin prisms predominantly formed, though isolated bipyramids were seen. X-ray diffraction was performed to investigate the role of tartrate in the formation of the different habits. The high purity and homogeneity of the thaumatin (thaumatin I) resulted in high resolution structures (up to 0.94 Å). We find that all bipyramids have a tetragonal unit cell, while the prisms are orthorhombic. The prisms never contain tartrate ions in the crystal structure, but the bipyramids can contain L-tartrate, D-tartrate or no tartrate, depending on how the crystals were produced, with L-tartrate being the most common. As the structure of the protein in the two habits is essentially identical, our work shows that the stereoisomerism of precipitants is an important factor in protein crystallization.

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