The function of a protein is encoded by its 3D structure. Protein crystallography is used as the major biophysical approach to investigate protein structure and function. A world class protein crystallography facility together with a high throughput protein expression facility for protein structure determination can be used to examine proteins of prime importance to human health. The recent advances in molecular medicine have led to an increase in the demand for structural information about proteins and, at the same time, an increase in the throughput of protein structure determination. As a primary manufacturer, Creative Biomart provides proteins of several sources, grades and formulations obtained by several methods for crystallography research applications.

Creative Biomart Product of Crystallography

Protein crystallization

Protein crystallization is a technique for the formation of protein crystals. It is an important step in protein crystallography. Protein crystallization is dependent on several factors such as the type of protein, type of precipitant, protein concentration, precipitant concentration, pH value, temperature, etc. Therefore, crystallographers experiment with many different combinations of these crystallization conditions when crystallizing proteins. Relatively large sized single crystals are useful for studying the structures and functions of proteins via an X-ray diffraction mechanism.

In conventional protein X-ray crystallography, a complete data set is ideally obtained from a single crystal, which typically requires a relatively large crystal that has successfully been cryo-cooled. Serial Crystallography takes the opposite approach: complete diffraction sets are assembled from a large number of individual diffraction frames acquired from small, single, randomly oriented crystals that are not cryo-protected. Complete coverage of the Ewald sphere is obtained by assembling individual diffraction frames into a single data set. The ideal crystals for serial crystallography are large enough and sufficiently defect free to diffract to high resolution, are produced in large quantity, and are sufficiently identical to facilitate merging of diffraction frames.

Protein crystallization techniques

In traditional crystallization methods, like microbatch, vapor diffusion, free interface diffusion, or microdialysis, a drop containing protein and reservoir is irreversibly quenched to a high supersaturation to facilitate crystallization.

In microbatch equal volumes of one part low ionic strength protein and one part precipitant solution are mixed and deposited under an oil film. As there is no connected reservoir, microbatch is often portrayed as a method in which the supersaturation does not change in time. Because of the low solubility of water in oil though, the protein solution dehydrates over periods of several weeks.

In vapor diffusion in turn, the drop containing protein and a reservoir are connected through a vapor bridge. The initial conditions of the protein containing drop are equal volumes of one part low ionic strength protein and one part reservoir solution, so initially the osmotic pressure of the drop is around half that of the reservoir. Water evaporates from the protein drop until the osmotic pressures of the reservoir and protein drop are equal, which means the protein drop approximately doubles the concentration of all its components. Typically, the increase in concentration of the protein solution from initial to final state takes 24 hours to occur.

In free interface diffusion as popularized by Salemme, protein solution and precipitant are loaded into a capillary, such that a concentration gradient can form along the capillary. Though often compromised by pipetting errors, gravity and movement, the gradient in concentration often results in a gradient in supersaturation along the capillary that often manifests itself through crystals of different size and density along the capillary.

In microdialysis the protein is loaded into a small dialysis chamber. A semipermeable dialysis membrane allows to gradually wash in precipitant, while retaining the protein inside the chamber. As this method allows to gradually wash detergents into or out of the protein sample, microdialysis is popular in crystalizing membrane proteins. For example, Efremov et al. used microdialysis to improve crystals from respiratory complex I, which were originally grown by vapor diffusion and that diffracted to ∼7 Å resolution, to ultimately diffract at 3.0 Å resolution.

Neither microbatch, vapor phase, nor free interface diffusion are reversible and each method scans one particular kinetic path in which supersaturation is steadily increased in time. While microdialysis allows for easy exchange of the precipitant solution and thus supersaturation could be decreased again in theory.

Protein crystallization factors

Salt concentration modulates protein solubility through generic electric charge screening, but also specific effects. At low salt concentrations protein surface charges are poorly screened, causing a net repulsion. Increased screening at medium salt concentrations results in higher protein solubility often referred to as “salting in”. At high ionic strength, protein and salt compete for solvent molecules and the net attraction between proteins becomes too high, ultimately causing precipitation of protein aggregates out of solution, also known as “salting out”. In addition, protein solubility can respond to salt type, as ions could adsorb onto the protein surface at specific sites or by modulating the dielectric constant of the solvent medium. Especially multivalent ions of the Hofmeister series are known to change the water structure and to strongly affect protein solubility.

The pH affects proton association and dissociation to surface exposed acidic and basic amino acid residues and thus the net charge of suspended protein. The pH value at which a protein has no net charge is known as the isoelectric point. At this isoelectric point the attractive polar interactions between protein and water and hence protein solubility are minimal, but solubility steeply increases at both lower and higher pH. Statistical analysis of successful crystallization conditions suggest that screening should focus on a range at or near the isoelectric point.

Polymers like polyethyleneglycol (PEG) are common additives in crystallization screens. Through depletion interaction, polymers exert an attractive force between protein molecules that arise from the difference in osmotic pressure between bulk fluid and the volume of fluid between the proteins from which the polymer has been excluded. Its magnitude depends on polymer concentration, while the range of the attraction is set by the polymers radius of gyration and polymer concentration.

Reference

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2. Chayen N E. Turning protein crystallisation from an art into a science[J]. Current opinion in structural biology, 2004, 14(5): 577-583.
3. Salemme F R. A free interface diffusion technique for the crystallization of proteins for X-ray crystallography[J]. Archives of Biochemistry and Biophysics, 1972, 151(2): 533-539.
4. Efremov R G, Sazanov L A. Structure of the membrane domain of respiratory complex I[J]. Nature, 2011, 476(7361): 414-420.
5. Zhang Y, Cremer P S. Interactions between macromolecules and ions: the Hofmeister series[J]. Current opinion in chemical biology, 2006, 10(6): 658-663.
6. Ruckenstein E, Shulgin I L. Effect of salts and organic additives on the solubility of proteins in aqueous solutions[J]. Advances in colloid and interface science, 2006, 123: 97-103.
7. Kantardjieff K A, Rupp B. Protein isoelectric point as a predictor for increased crystallization screening efficiency[J]. Bioinformatics, 2004, 20(14): 2162-2168.
8. Kulkarni A, Zukoski C. Depletion interactions and protein crystallization[J]. Journal of crystal growth, 2001, 232(1): 156-164.