Crystallization

Protein crystallography requires well ordered crystals of the sample suitable for diffraction of X-rays. Since proteins are usually large spherical or ellipsoidal objects with irregular surfaces, large crystals, wherein the protein is highly ordered in a three dimensional array, are quite difficult to obtain. The interactions of the macromolecule complex within a crystal are dependent on a plethora of parameters, such as the temperature, pH, nature of the solvent, precipitant and salts added, as well as the presence of ligands. Moreover, many proteins are composed of multiple functional domains with internal or terminal flexible regions. As now commonly believed and practiced, one usually attempts to remove all the flexible and non-functional parts and retains only the smallest functional domains to facilitate growth of crystals. In principle, this approach increases the probability of getting crystals because any flexible parts might inhibit the orderly packing of macromolecules in a crystalline array.

All these parameters may inhibit or support crystal growth. In addition, the conditions for crystal formation might be different from these optimal for crystal growth. Many crystallization conditions have to be tested during a crystallization experiment in order to obtain a condition which promotes growth of crystals suitable for X-ray diffraction analysis.

Crystals form when proteins are precipitated very slowly from supersaturated solutions. This thermodynamic driven process includes nucleation, crystal growth and growth termination. The most frequently used procedure for obtaining crystals is the vapour diffusion method[180].

In this thesis, the hanging drop vapour diffusion method was used. Therefore a buffered protein solution is mixed with precipitant solution from the reservoir in a 1:1 ratio on top of a cover slide. The reservoir contains a more concentrated precipitant solution and once the reaction chamber is sealed by turning the cover slide over and placing it on top of the chamber, slowly equilibrium between the reservoir and the hanging drop is reached. This optimally causes a saturation of the protein in the drop and if all conditions are right, protein crystals will occur in the drop.

3.4.1.1 Crystallizing a DNA polymerase in ternary structure

Crystallizing protein-DNA complexes is similar to crystallization of any macro- molecule. It depends on precipitant, ionic environment, pH and additives. DNA length is often the key factor that determines whether a protein-DNA complex crystallizes[181]. Since crystals are often grown at room temperature, one needs at least 7 bps to keep a DNA duplex stable at 20 °C. DNA sequences starting from the minimal length with increments of 1-2 bps can be screened for cocrystallzation.

Another variable is the sequence of the extra DNA that flanks the central essential sequence, particularly bases at the ends of DNA. It is often observed that DNA ends in crystals are packed against other DNA ends or protein molecules. Therefore, sticky ends, which contain single or double unpaired bases, are often employed so that the two ends of a single DNA are complementary. For example, one end has a 5’ protruding T and the other end has a 3’ overhanging A. Such DNA can polymerize in a head-to-tail fashion to form a repetitive linear array that potentially facilitates crystal growth[182]. To capture a DNA polymerase and substrate DNA binary complex, one only needs to mix the protein with a DNA that contains both a double-stranded region and a 5’ overhanging single stranded region. The junction between the double and single strand portions defines a specific site for polymerase to act[102]. For capturing a DNA polymerase, DNA substrate and incoming nucleotide in ternary complex, multiple approaches to stall the chemical reaction have been applied, which include:

Using a dideoxynucleotide triphosphate as an incoming nucleotide. A number of polymerases discriminate against dideoxynucleotide triphosphates, and these nucleotide analogs reduce the chemical reaction rate so that an enzyme-substrate complex can be captured[183, 184].

Using a 3’ dideoxy primer strand, which does not contain an hydroxyl nucleophile to form a covalent bond with an incoming nucleotide[100].

Replacing Mg2+_{, which is often essential for a chemical reaction, by Ca}2+_{, which}

enables the DNA and nucleotide association but is ineffective in facilitating catalysis[185].

Stabilizing protein-DNA complexes by covalent cross-linking. The basis for the design is the hypothesis that a protein α-helix tracks the DNA minor groove adjacent to the active site. By systematically replacing residues along that α-helix with Cys and modifying the DNA minor groove with a thiol group, a specific pair of modified protein and DNA can produce disulfide cross-linked complexes with high efficiency, while retaining the native conformation[186].

3.4.1.2 Crystal Seeding

If the concentration of a crystallizing protein is plotted against the concentration of the crystallizing agent, the resulting diagram divides the space into several areas depending on the physical state of the protein (Figure 3-14).

At very high concentrations of both protein and crystallizing agent, the protein precipitates as an amorphous material.

At lower concentrations, crystal nuclei may form, which can subsequently grow to diffracting crystals.

At still lower concentrations, nuclei will not form, so generally no crystals appear. However, if a nucleus or crystal is placed in such a solution, it will continue growing. This area, where crystals grow but no nucleation takes place, is referred to as the metastable zone.

At even lower concentrations the protein is completely soluble and nucleuses or crystals placed in such a solution dissolve.

It is often found that crystals grown in the metastable zone are better ordered and thus diffract better than crystals grown at higher concentrations. Also, sometimes it is difficult to obtain big enough crystals for diffraction in the nucleation zone, due to many but small crystals formed at these conditions. To grow crystals in the metastable zone, small crystals acting as nuclei, can be transferred into crystallization drops with lower protein and/or precipitant concentration. Crystal seeding can be performed as microseeding, by introducing only fragments of crushed crystals, or as macroseeding, by transferring an entire crystal to a new drop.

Figure 3-14. Crystallisation phase diagram. Schematic representation of a two-dimensional phase diagram, illustrating the change of protein molecules concentration against precipitating agent concentration. The concentration space is divided by the solubility curve into two areas corresponding to undersaturated and supersaturated state of a protein solution. The supersaturated area comprises of the metastable, nucleation and precipitation zones.