W0243

Protein Crystal Nucleation Kinetics: Is the Particle Pair Interaction Strength the Primary Determinant of Kinetics? C. Schall, R. Gosavi, S.Varanasi, V. Bhamidi, Dept. of Chemical Engineering, Univ. of Toledo, Toledo. OH.

Crystallization of biological molecules is the first step in growth of high quality crystals for structure determination, which involves the screening of a large sampling of conditions in search of suitable crystal formation conditions. Fundamental understanding of the phenomena of nucleation and crystallization can reduce the scope of conditions examined and provide insight into the mechanisms of nucleation/crystallization (and/or precipitation) of biological molecules. Our experimental data indicate that nucleation kinetics are primarily governed by the osmotic second virial coefficient, B₂₂, which is a measure of the strength of protein pair interactions. It has been well documented in the literature that (i) globular protein solubility is correlated with B₂₂ and (ii) the conditions conducive to crystallization fall into a defined range of values of B₂₂ termed the ‘crystallization slot’. The presence of a crystallization slot and our correlation of nucleation kinetics with B₂₂ indicates that the search for optimal crystallization conditions may, in large part, be a search for appropriate nucleation conditions. In our protein crystal nucleation studies, we examined the effects of precipitants and solubility enhancers on nucleation kinetics and solubility of hen egg white lysozyme. Nucleation kinetics were obtained by the method of initial rates. Nucleation rate (J) data were modeled using an empirical relation based on classical nucleation theory:

,

where A and B are constants, and C and C* are the bulk concentration and the equilibrium solubility of the protein, respectively. Within the framework of classical nucleation theory, the constant B, which partly accounts for the energy barrier to nucleation, is proportional to σ³, where σ is the crystal-fluid interfacial-energy. Based on simple fluid models, it can be argued that all three parameters A, B and C*, in the above equation are correlated with B₂₂, which lends support to our experimental observation that J is correlated to B₂₂. However, while the parameters A and C* are directly correlated with B₂₂, B correlates inversely with B22. Thus, substances, which are solubility-enhancers, could also promote nucleation kinetics via their ability to reduce the energy barrier to nucleation. These aspects would be addressed in the presentation.