Nonclassical Pathways of Protein Crystallization in the Presence of Multivalent Metal Ions

Using bovine beta-lactoglobulin as a model system, we have studied the crystallization pathways in the presence of the di- and trivalent salts ZnCl2 and YCl3. Previous work has shown that protein solutions undergo a reentrant condensation (RC) phase behavior in the presence of YCl3, i.e., a condensed phase occurs in between two boundary salt concentrations, c*< c**. In the presence of ZnCl2, c* also exists, but protein solutions with high salt concentrations do not become completely clear at higher protein concentrations (>20 mg/mL). We thus denote this diffuse transition as pseudo-c**. Small angle X-ray scattering measurements show that the effective interactions change from repulsion to attraction near c*, and attractive interactions dominate around pseudoc**. Solutions near c* and pseudo-c** provide the optimized conditions for growth of high quality protein single crystals but with different pathways. While crystal growth near c* follows the classical one-step nucleation pathway, crystallization around pseudo-c** (for zinc) or c** (for yttrium) follows a nonclassical process with an intermediate phase appearing before crystallization. Furthermore, the intermediate phases strongly depend on the crystallization temperature. Samples with high salt concentrations exhibit a typical transition temperature, Ttr, below which the solutions become turbid. When crystallizing near Ttr, the intermediate phase consists of protein clusters; below Ttr, the intermediate phase corresponds to macroscopic protein aggregates which can further relax into a dense liquid phase before crystallization. However, the experimental data cannot distinguish whether nucleation occurs within or outside of the intermediate phase. Possible scenarios are discussed based on the equilibrium phase behavior of colloidal systems with a short-range attraction. The crystallographic analysis of the resulting crystals shows that metal ions are an integral part of the crystal lattice. Both types of metal ions can create new protein contacts in the crystal lattice via bridging; however, yttrium creates more bridging contacts compared to zinc.