FUNDAMENTAL PROCESSES OF MACROMOLECULAR CRYSTALLIZATION

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To fully understand the behavior of biological macromolecules such as proteins and nucleic acids, we must know the three-dimensional arrangements of their constituent atoms. Complete structural information of this sort comes from X-ray and neutron diffraction studies, which require large, high quality crystals of the molecule under study. As a result of recent advances in diffraction methods, the bottleneck in structure determination has become the crystallization of the molecule of interest. Although a variety of techniques have been developed, some important classes of molecules have remained very difficult to crystallize; for example, whole immunoglobulin proteins and integral membrane proteins (e.g. channels, receptors, and energy transduction proteins). However, there has been very little systemic research on the fundamental processes of macromolecular crystallization, and thus no firm basis for developing guidelines for effective crystal growth. The goal is to fill this gap. The specific research aims are two-fold: to continue studies on crystallization of the model system, hen egg white lysozyme, and to extend results of this work to other proteins. Investigations will cover the three temporal stages of crystal growth: nucleation, postnucleation growth, and cessation of growth. I. Nucleation. The technique of quasi-elastic light scattering, previously applied to study nucleation in lysozyme, will be extended to other proteins to test its general effectiveness for the optimization of conditions for nucleation. The first experiments will focus on the difficult to crystallize immunoglobulins to determine whether the problem lies in the nucleation step or in later growth. II. Postnucleation Growth. Work will continue on the growth mechanism of tetragonal lysozyme crystals. A first aim is to test the hypothesis that two different mechanisms are involved in different regimes of supersaturation. A second concerns the effect on crystal growth of various protein impurities which may contaminate crystallization solutions. A third aim is to investigate whether crystal quality, as measured by the maximum resolution attainable in diffraction studies, is correlated with growth conditions and perhaps growth mechanism, thus establishing optimization guidelines. III. Cessation of Growth. It has been observed that protein crystals may cease to grow before they are large enough to be useful. The first requirement is to establish, under well-defined conditions, when cessation, or more generally reduction of growth rate, may occur. The next task is to investigate the cause of the reduction or cessation, in particular whether it is due to an accumulation of disorder as the crystal grows, a poisoning of the surface (e.g. by an impurity), or possible protein denaturation at the surface which inhibits further attachment.