Section 24.4.5. Reproducing published crystallization procedures

The BMCD contains the information needed to reproduce the crystallization conditions for a biological macromolecule reported in the literature. This is an activity performed by many laboratories engaged in protein engineering, rational drug design, protein stability and other studies of proteins whose structures have been previously determined. The crystallization of sequence variants, chemically modified derivatives, or ligand–biological macromolecule complexes can sometimes be considered problems that fit into this category. Usually, the reported crystallization conditions of the native macromolecule are the starting points for initiating the crystallization trials. The crystallization of the biological macromolecule may be simple to reproduce, but differences in the isolation and purification procedures, reagents, and crystallization methodology of different laboratories can dramatically influence the results. The crystallization conditions in the database should be considered a good starting point for the search or optimization that will require experiments that vary pH, macromolecule and reagent concentrations, and temperature, along with the crystallization method.

The crystals for one of the isozymes of glutathione S-transferase of rat liver grown from conditions reported in the literature (Sesay et al., 1987) are used to illustrate these points. The original crystallization conditions were for an enzyme isolated from rat liver (entries M0P3 and C13R). However, the enzyme used in the crystallization trials was cloned and expressed in Escherichia coli. The crystals of the natural enzyme were grown in 3 to 5 days from vapour-diffusion experiments at 4 °C, with droplets containing a protein concentration of 11.3 mg ml⁻¹, 0.46% β-octylglucoside, 30–37% saturated ammonium sulfate and 0.1 M phosphate buffer, pH 6.9 equilibrated against well solutions containing 60–74% ammonium sulfate.

The recombinant enzyme required an optimization of these conditions to produce large single crystals (Ji et al., 1994). The recombinant protein crystallized best at 4.0 °C, with droplets containing a protein concentration of 12 mg ml⁻¹, 0.2% β-octylglucoside, 20–25% saturated ammonium sulfate, 1 mM EDTA and 0.025 M TrisHCl, pH 8.0 equilibrated against well solutions containing 40–50% ammonium sulfate. Both crystallization protocols required the presence of 1 mM (9R,10R)-9-S-glutathionyl-10-hydroxy-9,10-dihydrophenanthrene, a product inhibitor. The recombinant enzyme, β-octylglucoside and ammonium concentrations were adjusted. The pH was varied, with the largest crystals being found at pH 8.0. Thus, TrisHCl was substituted for the phosphate buffer. EDTA was also included as an additive; its absence or presence did not affect the crystallization. Crystals of the recombinant enzyme grew within 5 to 10 days (Fig. 24.4.5.1).

Figure 24.4.5.1| top | pdf | Crystal of recombinant rat liver glutathione S-transferase (Ji et al., 1994) grown from optimized conditions based on the data in BMCD entries M0P3 and C13R.