Proteins play an essential role in nearly all biological processes. In many instances, that role can not be understood in detail without a knowledge of the three dimensional molecular structure of the protein. For example, if a drug is to be engineered to target a given protein, the protein structure is often the key to knowing what chemical functional groups to synthesize into the drug molecule. When single crystals of sufficient size and quality are available, the molecular structure of a protein can be determined by x- ray diffraction. The lack of quality crystals, however, is often the only obstacle standing in the way of determining the structure of a given protein. The objective of this proposal is a comprehensive series of experiments designed to establish the atomic scale physicochemical parameters controlling the rate of growth of the model protein, hen egg-white lysozyme. This protein has been chosen because: (1) It is available commercially with adequate purity. (2) Its solubility in water is known over wide ranges of temperature, pH, and concentrations of supporting electrolytes. (3) Its crystals grow rapidly and steadily to millimeter size and larger. In batch experiments, lysozyme crystals grow faster with increasing pH and ionic strength, while they grow slower with increasing temperature. The investigators propose to examine the effect of these variables on lysozyme crystals grown in evaporating hanging drops, where unlike batch experiments, the protein supersaturation is under control. They plan to use optical microscopy to determine the number of crystals and their average size as a function of supersaturation. They plan to analyze these data in terms of the Johnson-Meal-Avrami-Kolmogoro theory of nucleation and growth, which they have modified to be applicable to the hanging drop. The data will be used to determine the temperature, pH, and ionic strength dependence of the atomic scale nuclear ion and growth parameters that appear in the theory. By knowing how these parameters are determined by the solution conditions, one should be able to recommend procedures for enhancing the rate of growth of proteins that have so far resisted crystallization.
