Protein crystallization is an important tool to purify proteins as well as to demonstrate their chemical purity.
This process is essential for X-ray crystallography, a field which has contributed enormously to our understanding of atomic and molecular structure even at protein and nucleic acid level. These crystals help delineate the three-dimensional structure of complex macromolecules and their functions, as well as how they operate in the real world.
Principle
The crystallization of proteins is based on the careful combination of the supersaturated solution of the compound with precipitating or crystallization reagents under the right laboratory conditions to induce nucleation and growth of a protein crystal.
Procedure
The following steps are followed to crystallize a protein:
Protein Characterization
At least a few milligrams of the protein need to be purified and then characterized by circular dichroism (CD) spectroscopy to study protein structure and activity, differential scanning fluorometry, dynamic light scattering which ensures low polydispersity, or ultracentrifugation. Protein stability in the presence of various additives and ligands must also be studied. This step is essential for the success of crystallography.
Protein Crystallization
This process is dependent upon physical and chemical laws, and is the result of precipitating a supersaturated protein solution. The inability to use high temperatures to achieve supersaturation of proteins is overcome by using other factors such as different types of salt in buffer solution, or using co-factors to increase the solubility of the protein. Precipitants in common use include ammonium sulfate and polyethylene glycol. Commercial premade screening solutions are used to find out the kind of conditions which favors protein crystallization. These conditions are then optimized to enable the growth of large pure crystals which can be subjected to X-ray crystallography.
The most commonly used methods for protein crystallization include hanging drop and sitting drop, both used with vapor diffusion methods.
International Space Station Protein Crystal Growth Play
Difficulties with Protein Crystallization
The sample needs to be prepared and purified with care and solubilized in the right buffer environment to produce crystals. The utmost purity and monodispersity possible is the aim of sample preparation.
Stability is another primary concern. Storage must pre-empt any deterioration in terms of conformational change or denaturation, oligomerization or any such change before or during the process of crystal formation.
Supersaturation is then achieved using the right combination of reagents, pH of the buffer, the right temperature, and excipients or additives. The protein molecules are encouraged to associate in an orderly manner without precipitation or phase separation, or disorderly aggregation. Once nucleation is induced appropriately, with adequate numbers, size and quality, the environment must be tended to restrict further nucleation and promote controlled crystal growth. One grown, the crystals need to be protected against physical or chemical damage.
Control of the system is essential to keep it pure, fully specified, and unchanged, from beginning to end. This prevents the introduction of impurities into the growing crystal and ensures that laboratory conditions are reproducible.
Unfortunately, this process depends upon a whole host of biochemical, physical, and chemical factors. This makes standardization of protein crystallization a vexing task, with the necessity of understanding the right mix of chemicals, the behavior of the protein in various phases, and the nucleation as well as growth of crystals. Robotic systems to deal with these processes automatically and precisely, in nanoliter quantities, have been developed for larger laboratories, but are often too expensive for smaller setups.
Protein crystallization thus remains an art and science which is still under construction. The growth of a crystal whose unit is a macromolecule composed of thousands upon thousands of atoms in complex arrangements in primary, secondary and tertiary structures, with various degrees of freedom possible to each atom, is necessarily a task which is still guided by empirical knowledge so far rather than established theory.
References
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3943105/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1764865/ http://www.proteinstructures.com/Experimental/Experimental/crystallization-tools.html https://hamptonresearch.com/documents/growth_101/35.pdf https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182643/ http://www.xray.bioc.cam.ac.uk/xray_resources/whitepapers/xtal-in-action/node3.html
Further Reading