W0208

How Do Macromolecular Impurities Affect Protein Crystal Quality? C. L. Caylor, I. Dobrianov, K. D. Finkelstein, and R. E. Thorne, Cornell University, Ithaca, NY 14853

Typical growth solutions used in protein crystallization contain a variety of macromolecular impurities in concentrations of up to several molecular percent. These impurities can have dramatic effects on crystal nucleation and growth, and can lead to substantial crystal disorder. Previous studies have found that impurity concentrations within the crystals are usually orders of magnitude smaller than those present in solution. Vekilov et al. have observed enhanced salt incorporation in the core region of lysozyme crystals that correlates with solution impurity concentration, suggesting that impurity concentrations are larger in the core than in the bulk.

Our x-ray topography, mosaicity, and diffraction resolution measurements indicate that impurities cause crystal disorder primarily by creating defects in the early stages of growth. Lysozyme crystals grown from solutions prepared using high-purity commercial lysozyme are highly ordered, with featureless topographs and very narrow mosaic widths. Crystals grown from solutions intentionally contaminated with a variety of macromolecular impurities usually show profound disorder, particularly for larger impurity concentrations. However, crystals initially grown in uncontaminated solutions and then transferred while still small to contaminated solutions for the majority of their growth can be as perfect and well-ordered as crystals grown from uncontaminated solutions. Large, high-quality crystals can be obtained even for impurity concentrations an order of magnitude larger than those that cease to yield well-faceted crystals when growth occurs in a single solution. This suggests that the disorder is not due to bulk impurity incorporation or to impurity effects on growth kinetics, but is due to impurity-induced defects in the initial crystal nucleus that propagate and multiply as the crystal grows. These findings suggest a potential practical consequence: for some protein/impurity combinations, only very small quantities of highly purified protein--sufficient to produce seeds--need be prepared in order to grow high-quality crystals.