Protein crystallization is a highly demanding and time-consuming technique that constitutes a significant time constraint for basic research applications. Robotics allow high-throughput screening and optimization of crystallization conditions while minimizing the amount of protein necessary to conduct initial testing. However, the ability to distinguish a protein crystal from salt or amorphous material has not been automated, so each crystallization drop requires individual inspection.
The techniques presently used to identify protein crystals include:
- polarized light (birefringence)
- colorimetric staining
- crush test
- SDS-Page and WB
- In-situ x-ray diffraction
With the exception of polarized light, reliable only for large crystals, each method requires the crystallization drop be disturbed and/or destroyed during processing.
Tryptophan fluorescence
An increasingly popular method utilizes the intrinsic fluorescent properties of tryptophan. Tryptophan is excited with 280 nm UV light and the subsequent fluorescent emission captured between 300 -350 nm. One of the limiting factors for this method is the availability of UV compliant materials. In fact, many of the substrates commonly used for crystallization attenuate UV light at 280 nm. A comparison of the absorbance spectra of glass, film, and crystallization substrates for hanging drop and sitting drop crystallization provides a useful overview of the materials most suitable for this application
Method
Spectral properties of various commercially available media were assessed. A spectrophotometer was used to record absorbance spectra of a selection of commercially available crystallization substrates and films used to seal crystallization plates, in the range between 200 and 800 nm.
Conclusions
ProCrystals exhibit one of the highest UV transmission of all substrates at 280 nm, making them the ideal substrate for tryptophan-fluorescence-based identification of protein crystals.
Identification of crystallization conditions, by matrix screening, is usually followed by an optimization step. Initial crystals, in fact, are usually unsuitable for x-ray data collection due to their insufficient size or unfavorable morphologies that yield poor diffraction intensities. The resolution of an X-ray structure is directly correlated with the size and the quality of the crystalline samples; thus, refinement of conditions is often a critical component of crystal growth. Optimization of crystallization conditions requires the fine adjustment of the chemical and physical parameters that influence crystallization, such as pH, ionic strength, precipitant concentration, temperature, sample volume and overall methodology. This process is carried out manually, often using the hanging drop vapor diffusion method. When the initial screening is conducted with the same method, optimization of crystallization conditions is facilitated and larger well-formed crystals can rapidly be obtained. ProCrystals are compatible with high throughput crystallization techniques, allowing the hanging drop vapor diffusion method to be carried out in a 96 well format. ProCrystals can systematically increase the success rate of crystallization experiments and help obtain high-resolution macromolecular structures.
References
- Harindarpal S. Gill et al. “Evaluating the efficacy of tryptophan fluorescence and absorbance as a selection tool for identifying protein crystals. Acta Cryst. (2010). F66, 364-372
- McPherson A. et al. “Optimization of crystallization conditions for biological macromolecules.” Acta Crystallogr. Struct Biol Commun. 2014 Nov; 70 1445-67M
