This thesis investigates improvements in protein crystal quality for X-ray crystallography. Knowledge of protein structure on an atomic level is critical for the fundamental understanding of protein function, and for structure based pharmaceutical drug design. X-ray crystallography is currently the dominant method of protein structure determination. The resolution of the protein structure determined via X-ray crystallography is highly dependent upon the quality of the diffracting crystal. Despite the importance of crystal quality for structure determination, systematic studies of protein crystallization have been largely ignored until fairly recently. The work presented here focuses on the development of a rational approach to protein crystallization. This approach consists of developing a basic kinetic and thermodynamic understanding of the protein solvent system. Information gleaned from this characterization work is then used to calculate a crystal growth algorithm for the system. The crystal growth algorithms use either temperature or precipitant concentration to dynamically control protein solubility during crystallization. Dynamic solubility control is advantages as it allows for the use of much lower supersaturation levels and therefore greatly reduced rates of crystal nucleation and growth.; Previous work utilized temperature control algorithms to grow crystals of lysozyme in NaCl. However, the use of temperature control is precluded in proteins that do not have a temperature dependent solubility. This work addresses this difficulty in two ways. First, low ionic strength solvent systems (NaNO3 and NaSCN) were used to enhance the temperature sensitivity of lysozyme. Large well-formed crystals were grown in both of these solvents using temperature control algorithms. Second, lysozyme crystals were grown via precipitant control algorithms. Here dynamic control is provided by slowly increasing the precipitant concentration isothermally. For these experiments the lysozyme/NaSCN system was employed to facilitate comparison between temperature and precipitant control. Large well-formed crystals were grown using a variety of precipitant control profiles. In terms of size and overall quality of crystals produced temperature appears to be superior for this system. However both types of algorithm produced crystals greatly superior to those grown without dynamic control. Development of precipitant control should allow these methodologies to be applied to a wide variety of systems.
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