Two-photon fluorescence imaging of impurity distributions in protein crystals

Macromolecular impurities present in solution have profound effects on the growth and quality of protein crystals used for x-ray structure determinations. We have imaged the three-dimensional distribution of ovotransferrin impurities in crystals of the protein hen egg white lysozyme (HEWL) using two-photon excitation fluorescence microscopy. Impurity concentrations differ between the two types of growth sectors present in tetragonal HEWL crystals and impurities preferentially incorporate along the boundaries between growth sectors. Cracked crystals show large impurity-rich cores that are not observed in uncracked crystals. These nonuniform impurity distributions provide insight into how and why impurities affect crystal quality. Our results have implications for crystal growth and for protein purification.