| Crystallization of biological molecules is the first step in growth of high quality crystals for structure determination. Formation of stable crystals of proteins and protein/nucleic acid complexes from solution is dominated by the kinetics of nucleation of these clusters. In order to obtain a fundamental understanding of nucleation kinetics and develop and test predictive models for this process, a critical amount of kinetic data must be collected and key solution parameters need to be identified.; Given the sparse amount of nucleation kinetic data available, the nucleation kinetics of tetragonal hen egg-white lysozyme, HEWL, were measured by the method of initial rates at various solution conditions. At 4°C, 0.1 M sodium acetate buffer at pH 4.5 and 5 to 7% (w/v) NaCl, the solid phase formed contained a powdery material instead of the expected tetragonal crystals. This powdery material was found to have a regular needle like structure when examined using optical microscopy. Powder diffraction data from these needles was inconsistent with the experimental powder diffraction data from tetragonal lysozyme crystals. It is possible that at high salt and protein concentrations liquid-liquid separation occurred and yielded a crystal polymorph.; Nucleation kinetic data were modeled using an empirical kinetic expression based on the classical nucleation theory. The nucleation rate data were split into two regions: heterogeneous nucleation region at lower protein concentrations and homogeneous nucleation region at higher protein concentrations. The model parameters A and B were estimated separately in individual regions. In both regions, parameter B was fairly independent of ionic strength. However, in the homogeneous range, the pre-exponential factor, A, increased monotonically with salt concentration and correlated with trends observed in osmotic second viral coefficients ( B22) and solubility.; It was also found that, for HEWL/NaCl system, for a given protein concentration (above equilibrium solubility) equal solubility conditions produce equal nucleation kinetics, even when the solution conditions such as pH, ionic strength and temperature are different. This observation, if shown to be valid for all protein systems, could have far reaching consequences, as it will significantly narrow the spectrum of parameters to be explored to identify optimal nucleation and crystallization conditions. |
