X-ray Crystallography

Growth of Protein Crystals.

Precipitants were used to grow proteins crystals by precipitation from aqueous solutions. There are diverse precipitants, including salts, organic solvents and polymers (e.g. PEG) and combinations of various types of precipitants can be used. Other criterion also influence crystal growth, such as precipitant

concentration, pH and temperature (Boistelle and Astier 1988). In order to find appropriate conditions for crystallisation of a protein, screening was achieved over a range of different conditions to see when crystals were formed and after that optimisation of those conditions was usually needed to improve the crystals.

The procedure of growing crystals usually involves addition of the precipitants to a aqueous solution of the protein in a concentration just below what is needed for precipitation. After that, the water is evaporated slowly until precipitating conditions was reached. The method used for crystallisation was vapour diffusion. This method is based on the technique described by Unge (1999). A droplet of purified protein solution mixed with a crystallisation solution is deposited onto a cover glass. The crystallisation solution often consists of buffer, salt and precipitant. The cover is suspended above a reservoir of crystallisation solution. Due to concentration gradient, the water from the drop evaporates towards the reservoir, thus lowering the volume of the drop and increasing the protein concentration. This will result in supersaturation of the protein solution and crystals will start to form (Unge 1999). Vapour diffusion can be performed in different ways, for example hanging drop and sitting drop, as shown in Figure 2.3.

Figure 2.3: Different vapour diffusion system. A) Hanging drop and B) sitting drop setups. Adapted from Unge (1999).

Co-Crystallisation.

Protein-ligand interactions can also be studied with X-ray crystallography. For this, crystals of protein-ligand complexes are needed. There are different ways of achieving this, including soaking and co-crystallisation. These two methods were used (Hassell et al. 2007).

Soaking: This method implicates transferring the protein crystals into crystallisation solution that contains the ligand. The ligand can diffuse through water channels in the crystal and thereby reach the active site. A requirement for this method is that the protein crystals are functional for binding ligands. This can vary for different ligands and the soaking process can be affected by solubility, size and shape of the ligand.

Co-crystallisation: The ligand and protein are mixed and crystallised together. This method is more time-consuming and requires more protein. The crystal structure can differ with different ligands and might not be the same as for crystals of the protein without ligand, and requires a new screening to achieve crystals.

Crystal Analysis.

Hanging drop setups were prepared manually using 24-well tissue plates as described in Chapter 3. However, sitting drop trays were set up using Honeybee® 963 and Mosquito® LCP3 _{crystallisation robots to screen 96◊1 mL}

trays. For each ligand (CP06 and CP67) three 96-condition crystallisation screens were used; JCSG+, PACT and Morpheus purchased from Molecular Dimensions Ltd. (Suffolk, UK). Crystallisation screening was performed using reservoir volumes of 50 µL, and 2 µL sitting drops comprising 1 µL of protein

solution and 1 µL of reservoir solution. The six trays were stored in a constant

temperature room at 18¶C and inspected daily using an Olympus SZ4045TR-PT microscope.

In both setups, generated crystals were tested with X-rays4_{. Crystals that}

diffracted to better than 4 Å resolution were taken to the the Diamond Synchrotron Source in Oxford. Model building for the crystals, that were tested so far at the synchrotron, was done by molecular replacement, as we already

3_{Dr Dean Rea (School of Life Sciences, University of Warwick) set up sitting drop screening}

using Mosquito LCP crystallisation robot.

4_{Dr Karen Ruane (School of Life Sciences, University of Warwick) tested crystals for}

diffraction, collected the data at Diamond Synchrotron Source in Oxford and solved the structure.

Protein Preparation for NMR

Spectroscopy and X-ray

Crystallography Studies

3.1 Introduction.

Due to its wide use in cancer therapy, there has been a massive need for high yield expression of CPG2. The first goal of this work was development of an

E. coli-based protocol for overexpression of CPG2 in a soluble form. E. coli

was a more attractive alternative to Pseudomonas sp. RS-16, previously used to produce CPG2 for crystallography studies, as it offers shorter culturing time, fast high density cultivation and easier genetic manipulation than Pseudomonas, the natural CPG2 source (Rowsell et al. 1997). Because Pseudomonas and E. coli

strain RS-16 was codon-optimised and cloned into a pET28a vector for maximum expression in BL21 (DE3) E. coli strain (Goda et al. 2009). In spite of all these qualities, expression of CPG2 with E. coli as the host resulted in high yields of the protein, but in its insoluble form (Goda et al. 2009). Such proteins, forming dense aggregates (i.e. inclusion bodies), are frequently improperly folded and thus biologically inactive (Singh and Panda 2005).

In the context of this project, it was vital to express high-yields of CPG2 in its soluble and active form in order to preserve the integrity of the protein fold for future structural studies.