Protein Crystallization

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 scratch is the need for a large number of experiments exploring the various conditions that are necessary for successful crystal growth. However, it is tedious to set up and screen so many different experimental conditions. Thus, high through-put methods exist to help streamline this process. There are numerous kits available to 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. Another method that has proven useful in setting up a vast number of crystallizations involves robotics. What would otherwise be slow and error-prone when carried out by a human can be accomplished efficiently and accurately with an automated system. Click here to see a movie of a crystallization robot in action (http://www.syrrx.com/technology/index.htm) . 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 (Eurekalert , 2000).

Links

A photo gallery of protein crystals.

NASA's protein crystal growth home page. (A microgravity environment is apparently conducive to successful crystal growth.)

Protein Crystallization and Dumb Luck. (An essay on the haphazard side of protein crystallization by Bob Cudney.)

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