Contaminant inclusion into protein crystals analyzed by electrospray mass spectrometry and X-ray crystallography.

The inclusion of protein contaminants into crystals of turkey egg white lysozyme (TEWL) was investigated by electrospray mass spectrometry of the dissolved crystals. The results show that significant amounts of the structurally related contaminant hen egg white lysozyme (HEWL) are included in the crystals of TEWL. The structurally unrelated contaminant RNAse A, on the other hand, is not included. The X-ray diffraction data statistics of a hybrid TEWL/HEWL crystal and an uncontaminated TEWL crystal were of similar quality. This indicates that, even though the crystals contain much higher levels of the contaminant than one would have expected after a recrystallization experiment, they are still suitable for X-ray diffraction experiments. However, attempts to detect the presence of the contaminant in the crystal by crystallographic structure refinement did not yield conclusive results.

Contaminant effects on protein crystal morphology in different growth environments.

Contaminant effects on the morphology of turkey egg-white lysozyme (TEWL) crystals grown in ungelled and in gelled growth media have been investigated. The latter may serve as a model system for future microgravity experiments. Hen egg-white lysozyme (HEWL) was added as the contaminant at levels ranging from 4 up to 70%(w/w) (total protein). Morphology measurements indicate a contaminant effect leading to a shortening along the c axis of the crystal. This shortening effect depends on the contaminant concentration. It is attenuated and varies more regularly in gelled than in ungelled growth media. The specificity of the HEWL contaminant effect was verified by addition of ribonuclease A, which did not influence crystal morphology. Contaminant inclusion into the growing TEWL crystals could be calculated directly from equilibrium protein concentration measurements. The level of HEWL inclusion is closely related to the concentration of HEWL in the growth solutions. The specificity of the observed effect as well as the differences between the two growth media are discussed.

The effects of filtration on protein nucleation in different growth media.

Filtration effects of turkey egg white lysozyme solution (TEWL) prior to subjecting it to crystallization conditions are investigated. Filtering TEWL solution and crystallizing it in ungelled media significantly decreased the number of conditions yielding crystals. This decrease dependent on the membrane cut-off used for filtration. From this, the postulated factors aiding in nucleation are estimated to be 0.17 microns in diameter. The existence of these factors was verified by the procedure of reversed filtration: filtered solutions passed through their inverted filter membrane a second time lead to improved crystallization results. The effect of aging of the TEWL solution prior to subjecting it to ungelled crystallization conditions was also verified. We did not find any time-dependent change in the size or the number of crystals per drop. Repeating the filtration experiments in agarose-gelled crystallization media showed that the influence of filtration on the crystallization outcome was significantly diminished. Far better crystallization results were obtained compared to ungelled media. However, there is a certain aging effect linked to filtration in gelled media. Different crystallization results were obtained depending on whether filtration was performed before or after aging and subsequent crystallization. This suggests a secondary time-dependent effect.