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Protein crystallization

Protein crystallization is the process of formation of a protein crystal. In the process, proteins will dissolve in an aqueous environment, and be recrystallized by different methods. Such as, Vapor diffusion, Microbatch, Microdialysis, Free-interface diffusion, etc. Some parameters including pH, temperature, ionic strength in the crystallization solution and other factors will influence the result.

Based on the crystal, the determination of protein structure can be achieved traditionally by utilizing X-Ray Diffraction (XRD). Alternatively, cryo-electron microscopy (cryo-EM) and nuclear magnetic resonance (NMR) could also be used for protein structure determination. The structural chemistry of biological macromolecules, such as proteins, is significant due to the need of structural analysis in biochemistry and translational medicine.

Development of protein crystallization

In 1840, Friedrich Ludwig Hünefeld accidentally discovered the formation of crystalline material in samples of the earthworm blood held under two glass slides and occasionally observed small plate‐like crystals in desiccated swine or human blood samples. These crystals ware named as ‘haemoglobin’, by Felix Hoppe‐Seyler in 1864.

In 1934, John Desmond Bernal and his student Dorothy Hodgkin discovered that protein crystals surrounded by their mother liquor gave better diffraction patterns than dried crystals. Using pepsin, they were the first to discern the diffraction pattern of a wet, globular protein. Prior to Bernal and Hodgkin, protein crystallography had only been performed in dry conditions with inconsistent and unreliable results.

In 1958, the structure of myoglobin, determined by X-ray crystallography, was first reported by John Kendrew.Kendrew shared the 1962 Nobel Prize in Chemistry with Max Perutz for this discovery

Methods of protein crystallization[edit]

Vapor diffusion[edit]

Three methods of preparing crystals, A: Hanging drop. B: Sitting drop. C: Microdialysis Vapor diffusion is the most commonly employed method of protein crystallization. In this method, a droplet containing purified protein, buffer, and precipitant are allowed to equilibrate with a larger reservoir containing similar buffers and precipitants in higher concentrations. Initially, the droplet of protein solution contains comparatively low precipitant and protein concentrations, but as the drop and reservoir equilibrate, the precipitant and protein concentrations increase in the drop. If the appropriate crystallization solutions are used for a given protein, crystal growth will occur in the drop. This method is used because it allows for gentle and gradual changes in concentration of protein and precipitant concentration, which aid in the growth of large and well-ordered crystals.

Vapor diffusion can be performed in either hanging-drop or sitting-drop format. Hanging-drop apparatus involve a drop of protein solution placed on an inverted cover slip, which is then suspended above the reservoir. Sitting-drop crystallization apparatus place the drop on a pedestal that is separated from the reservoir. Both of these methods require sealing of the environment so that equilibration between the drop and reservoir can occur.

Microbatch[edit]

A microbatch usually involves immersing a very small volume of protein droplets in oil (as little as 1 µl). The reason that oil is required is because such low volume of protein solution is used and therefore evaporation must be inhibited to carry out the experiment aqueously. Although there are various oils that can be used, the two most common sealing agent are paraffin oils (described by Chayen et al.) and silicon oils (described by D’Arcy). There are also other methods for Microbatching that don't use a liquid sealing agent and instead require a scientist to quickly place a film or some tape on a welled plate after placing the drop in the well.

Besides the very limited amounts of sample needed, this method also has as a further advantage that the samples are protected from airborne contamination, as they are never exposed to the air during the experiment.

Microdialysis[edit]

Microdialysis takes advantage of a semi-permeable membrane, across which small molecules and ions can pass, while proteins and large polymers cannot cross. By establishing a gradient of solute concentration across the membrane and allowing the system to progress toward equilibrium, the system can slowly move toward supersaturation, at which point protein crystals may form.

Microdialysis can produce crystals by salting out, employing high concentrations of salt or other small membrane-permeable compounds that decrease the solubility of the protein. Very occasionally, some proteins can be crystallized by dialysis salting in, by dialyzing against pure water, removing solutes, driving self-association and crystallization.

Free-interface diffusion[edit]

This technique brings together protein and precipitation solutions without premixing them, but instead, injecting them through either sides of a channel, allowing equilibrium through diffusion. The two solutions come into contact in a reagent chamber, both at their maximum concentrations, initiating spontaneous nucleation. As the system comes into equilibrium, the level of supersaturation decreases, favouring crystal growth.

Technologies that assist with protein crystallization

High throughput crystallization screening

High through-put methods exist to help streamline the large number of experiments required to explore the various conditions that are necessary for successful crystal growth. There are numerous commercials kits available for order which apply preassembled ingredients in systems guaranteed to produce successful crystallization. Using such a kit, a scientist avoids the hassle of purifying a protein and determining the appropriate crystallization conditions.

Liquid-handling robots can be used to set up and automate large number of crystallization experiments simultaneously. What would otherwise be slow and potentially error-prone process carried out by a human can be accomplished efficiently and accurately with an automated system. Robotic crystallization systems use the same components described above, but carry out each step of the procedure quickly and with a large number of replicates. Each experiment utilizes tiny amounts of solution, and the advantage of the smaller size is two-fold: the smaller sample sizes not only cut-down on expenditure of purified protein, but smaller amounts of solution lead to quicker crystallizations. Each experiment is monitored by a camera which detects crystal growth.

Protein engineering

Techniques of molecular biology, especially molecular cloning, recombinant protein expression, and site-directed mutagenesis can be employed to engineer and produce proteins with increased propensity to crystallize, or can even direct polymorph selection during protein crystallization . Frequently, problematic cysteine residues can be replaced by alanine to avoid disulfide-mediated aggregation, and residues such as lysine, glutamate, and glutamine can be changed to alanine to reduce intrinsic protein flexibility, which can hinder crystallization.

Applications of protein crystallization

Protein crystallization is required for structural analysis by X-ray diffraction, neutron diffraction, and some techniques of electron microscopy. These techniques can be used to determine the molecular structure of the protein. For a better part of the 20th century, progress in determining protein structure was slow due to the difficulty inherent in crystallizing proteins. When the Protein Data Bank was founded in 1971, it contained only seven structures. Since then, the pace at which protein structures are being discovered has grown exponentially, with the PDB surpassing 20,000 structures in 2003, and containing over 100,000 as of 2014.

Crystallization of proteins can also be useful in the formulation of proteins for pharmaceutical purposes.