4.3.1 Basics and principles in crystallization of macromolecules
Crystallization of a protein depends on many different factors. Variation of pH, con-centration and kind of buffer, concon-centration and kind of salt, temperature, dielectric constant, protein concentration and many more can decide on whether crystals grow or not. A phase diagram as shown in Fig.4.5 can visualize the influence of e.g.the salt concentration and the protein concentration on formation of crystals. In this diagram, the concentration of salt and protein are varied, while all other parame-ters are kept constant. The area of undersaturation is separated by the solubility curve from the area of supersaturation. If the concentration of the protein is so low that the solution is undersaturated, the protein cannot crystallize. Supersaturation is essential for the formation of crystals. The supersaturation zone can be subdi-vided into three zones. In the precipitation zone, the protein will precipitate as an amorphous solid, but not in crystalline form. In the nucleation zone, the protein nucleates in form of many small crystals. It can happen that saturation is reached but no crystal big enough for an X-ray diffraction measurement has formed. One has then the possibility to put a small crystal in a protein solution with a con-centration that corresponds to conditions of the metastable zone, as in this zone crystallization may only happen whene.g. seeds of small crystals are introduced to this solution. Crystals can grow under these conditions slow enough to form few big crystals suitable for X-ray diffraction without nucleation of new crystals.[75]
Figure 4.5: Schematic phase diagram for the crystallization of a protein.
4.3.1.1 Hanging drop vapor diffusion method
By the hanging drop vapor diffusion method the peptide solution is mixed in a drop on a siliconized cover slide with the reagent solution, which consists of salt, buffer, precipitant or other additives. The drop is equilibrated against a solution containing the reagents. As the concentration of the reagents in the drop is much lower than in the solution an equilibrium is reached by evaporation of water which moves from the drop into the solution (see Fig. 4.6). This leads to a supersaturation of the protein in the drop, which hopefully results in crystallization of the protein. This method can only be used if the drop is not too big as otherwise it would drop into the reagent solution instead of being equilibrated.
Figure 4.6: Hanging drop vapor diffusion crystallization.
4.3.1.2 Sitting drop vapor diffusion method
The sitting drop vapor diffusion method is similar to the hanging drop vapor diffusion method. Here again, a solution containing the peptide and the reagent solution are
equilibrated against each other (Fig. 4.7). As the drop is sitting on a bridge this method is suitable especially for large drops and drops containing reagents with a low surface tension.
Figure 4.7: Sitting drop vapor diffusion crystallization.
4.3.1.3 Cryocrystallography
When the measurement is performed at room temperature there are several problems that can occur. One is that radiation damage can destroy the crystal before the measurement can be finished successfully. Radiation damage occurs by primary interaction between the beam and the molecules in the crystal. The energy of the beam produces heat and thus increases the thermal vibration of the molecules, and this energy is also sufficient to break certain bonds in the molecules. Radicals that are produced in this way can then further diffuse through the crystal and cause even more damage.[76, 77] When the crystal is cooled down to 100 K the problem of radiation damage is less distinct. But as proteins often contain a lot of water care must be taken when the crystal is cooled down. It must be avoided that water freezes to ice and thus cracks the crystal. This can be achieved by soaking the crystal in an anti-freeze agent (cryoprotectant) and flash-cooling it down to 100 K.[78, 79] The anti-freeze agent prevents the water from freezing to ordered ice but water forms a vitreous glass, instead. When the molecules in the protein crystal are frozen vibration is reduced which allows the crystal to diffract to higher resolution. Also the degree of disorder (at least of dynamic disorder) is reduced at lower temperatures.
4.3.2 Crystallization conditions for A2
Crystallization conditions for A2 were found by screening with theJena Bioscience[80]
JBScreen Classic 8 when the conditions of the two neighboring wells D4 (60 % w/v ethanol, 1.5 % w/v PEG 6000, 0.05 M sodium acetate) and D5 (60 % w/v ethanol, 0.10 M sodium chloride) were accidentally mixed. As the concentration of the result-ing solution was not exactly known for this reason, a 24 well plate was set up with
varying conditions for ethanol, PEG 6000, sodium acetate and sodium chloride. The composition which yielded the best crystals was a reservoir mixture of 60 % ethanol, 0.75 % PEG 6000, 0.025 M sodium acetate and 0.05 M sodium chloride. For the crystallization the peptide was dissolved in 0.02 M TRIS of pH 8.2 with a concentra-tion of 7 mg/ml. For the drop 2µl of peptide were mixed with 2µl of the reservoir solution. Crystals grew within one week to a size of 0.125×0.075×0.025mm3 at room temperature by using the sitting drop vapor diffusion method. A crystal of A2 is shown in Fig. 4.8. The crystals were soaked for a few seconds in a solution containing 15 % 2,3-butanediol as a cryoprotectant. Subsequently, the crystal was flash frozen in liquid nitrogen and transferred quickly to the diffractometer.
Figure 4.8: Crystal of labyrinthopeptin A2.
X-ray data were collected on a Bruker three circle diffractometer equipped with a rotating anode with mirror-monochromated Cu-Kα radiation (λ = 1.54178˚A).
Intensities were measured with a SMART 6000 detector to a resolution of 1 ˚A.