Protein Crystallization

Background:
Proteins, like many molecules, can be prompted to form crystals when placed in the appropriate conditions. In order to crystallize a protein, the purified protein undergoes slow precipitation from an aqueous solution. As a result, individual protein molecules align themselves in a repeating series of "unit cells" by adopting a consistent orientation. The crystalline “lattice” (Fig. 1) that forms is held together by noncovalent interactions (Rhodes, 1993). The importance of protein crystallization is that it serves as the basis for X-ray crystallography, wherein a crystallized protein is used to determine the protein’s three-dimensional structure via X-ray diffraction. In 1934, Bernal and Hodgkin discovered that protein crystals surrounded by their mother liquor gave better diffraction patterns than dried crystals. Using pepsin (Fig. 2), 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 (Tulinsky, 1999).
Initial progress was slow in discerning protein shapes, due to the difficulty inherent in crystallizing proteins. When the Protein Data Bank ( ) was founded in 1971, it contained only seven structures (Berman, et al., 2000). Since then, the pace at which protein structures are being discovered has grown exponentially, with the PDB currently containing over 20,000 structures (PDB, 2003). Our ability to crystallize proteins has obviously improved, and this web page highlights a few of the methods currently available.Figure 2. Three-dimensional structure of porcine pepsin. Pepsin was the first globular protein crystal used successfully in x-ray diffraction. Image courtesy of Protein Databank (ID: 3PSG; PDB , 2003).Overview:The goal of crystallization is usually to produce a well-ordered crystal that is lacking in contaminants and large enough to provide a diffraction pattern when hit with x-ray. This diffraction pattern can then be analyzed to discern the protein’s three-dimensional structure. Protein crystallization is inherently difficult because of the fragile nature of protein crystals. Proteins have irregularly shaped surfaces, which results in the formation of large channels within any protein crystal. Therefore, the noncovalent bonds that hold together the lattice must often be formed through several layers of solvent molecules (Rhodes, 1993). In addition to overcoming the inherent fragility of protein crystals, the successful production of x-ray worthy crystals is dependent upon a number of environmental factors because so much variation exists among proteins, with each individual requiring unique conditions for successful crystallization. Therefore, attempting to crystallize a protein without a proven protocol can be very tedious . Some factors that require consideration are protein purity, pH, concentration of protein, temperature, and precipitants. In order for sufficient homogeneity, the protein should usually be at least 97% pure. pH conditions are also very important, as different pH’s can result in different packing orientations. Buffers, such as Tris-HCl, are often necessary for the maintenance of a particular pH (Branden and Tooze, 1999). Precipitants, such as ammonium sulfate or polyethylene glycol, are compounds that cause the protein to precipitate out of solution (Rhodes, 1993).Vapor Diffusion:Two of the most commonly used methods for protein crystallization fall under the category of vapor diffusion. These are known as the hanging drop and sitting drop methods. Both entail a droplet containing purified protein, buffer, and precipitant being allowed to equilibrate with a larger reservoir containing similar buffers and precipitants in higher concentrations. Initially, the droplet of protein solution contains an insufficient concentration of precipitant for crystallization, but as water vaporizes from the drop and transfers to the reservoir, the precipitant concentration increases to a level optimal for crystallization. Since the system is in equilibrium, these optimum conditions are maintained until the crystallization is complete (Rhodes, 1993; McRee, 1993).Simply put, the hanging drop method differs from the sitting drop method in the vertical orientation of the protein solution drop within the system. It is important to mention that both methods require a closed system, that is, the system must be sealed off from the outside using an airtight container or high-vacuum grease between glass surfaces. Figures 3 and 4 depict the hanging drop and sitting drop systems, respectively (Rhodes, 1993; McRee, 1993).High Through-Put Methods:One unavoidable aspect of crystallizing a protein from scra