Structure determination of biomolecules is essential to an understanding of structure function relationships and to the rational development of strategies for disease treatment and pharmaceutical development. Many biomolecules of interest are proteins which can vary in size from 10 to 500 kilodaltons. X-ray crystallography is the technique normally employed for structure determination of proteins larger than 20 kilodaltons. In order to determine the structure using x-ray crystallography, a crystal of suitable size must first be formed (minimum of about 0.1 mm in size). Crystallization can be modelled as two distinct processes; consisting of nucleation and crystal growth. The focus of this research is on the first step in the crystallization process, formation of crystal nuclei. The goal of this research is to examine the effect of the system properties on nucleation kinetics of protein crystals. This research will have the following specific objectives: Use calorimetry to monitor nucleation and crystallization kinetics. Isothermal calorimetry can be used to study the kinetics of slow reactions. Protein crystallization kinetics are slow enough to be monitored by calorimetry. Variable speed agitation is available on sensitive isothermal solution calorimeters. Rate of addition of components to the crystallization cell can also be varied with these calorimeters. Examine the role of mixing in nucleation of protein crystals. Does mixing increase or decrease nucleation kinetics in protein solutions? Is it best to agitate vigorously when mixing protein solutions and precipitants to reduce regions of high protein or precipitant concentration? It is hypothesized that the initial mixing of protein and precipitant solutions will have a significant effect on protein crystal nucleation and subsequent crystal growth. What is the dominant mechanism of nucleation in protein systems? Nucleation can occur through several mechanisms which can be broadly termed as primary or secondary nucleation. In order to predict the nucleation event, the system properties which control nucleation rates must be identified. The relationship between supersaturation and nucleation rates can give insights into the dominant nucleation mechanism. Properties of the macromolecule in solution, such as hydrodynamic radius and diffusitvity are also expected to influence nucleation rates.
