Thaumatin, an intensely sweet protein, crystallizes rapidly in the presence of tartrate ions. The ease with which crystals form has led to the use of thaumatin over the past decade as a model system for the study of protein crystallization. The available data on the solubility of this protein, however, are inconsistent. We have purified thaumatin and determined its solubility with the L and D enantiomers of the tartrate ion. We find that the crystal habit and solubility are significantly different for the two precipitants: the solubility increases with temperature in L-tartrate, while it decreases with temperature in D-tartrate. Our results suggest that the chirality of precipitants is an important factor that should be controlled when determining the solubility of proteins.

A major challenge in structural biology is to produce high-quality protein crystals for X-ray diffraction. Currently, proteins are crystallized by trial and error, often in multicomponent solutions with chiral precipitants. As proteins are chiral molecules, we hypothesized that the chirality of the precipitants may affect crystallogenesis. To test this hypothesis, we crystallized thaumatin, an intensely sweet globular protein, with the three stereoisomers (l-, d-, and meso-) of tartaric acid. We find three different crystal habits and crystal packings; the three stereoisomers interact with the protein at different sites. All three precipitants produce high-quality crystals from which atomic resolution (∼1 Å) structures were obtained. Our findings suggest that stereospecific interactions with precipitants are important in protein crystal formation and should be controlled when crystallizing proteins for structure determination.

ABSTRACT:Thaumatin is frequently used as a model protein in crystallization studies because it rapidly forms crystals in the presence of tartrate ions. The thermodynamic and kinetic properties of thaumatin crystals have been studied for almost 10 years, and the results are contradictory. Here we show that by using a homogeneous preparation of thaumatin and controlling the stereochemistry of the tartrate precipitant, it is possible to achieve consistent results for the protein solubility. To understand the role of protein impurities in the crystallization of thaumatin, we examined two commercial sources of the protein and characterized the heterogeneities therein. To examine the effect of precipitant stereochemistry, we crystallized thaumatin with L, D, and DL (racemic) tartrate ions. We suggest that the inconsistencies among previous results stem in part from the different behavior of thaumatin with the L and D enantiomers: the solubility of thaumatin crystals increases with temperature in L-tartrate, whereas it decreases with temperature in D-tartrate. Our results demonstrate the importance of using pure protein and stereochemically pure precipitants in the crystallization of proteins.

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