Crystallisation of ADPGK

Protein samples for crystallisation were prepared as described in section 2.6.1, ideally ensuring that ADPGK was of a purity of over 90% as assessed by SDS-PAGE. Potential aggregates were removed by filtration or centrifugation directly prior to crystallisation. A large number of crystallisation trials were set up for ADPGK. The strategy used for this work was mostly facilitated by access to a Mosquito pipetting robot (TTP Labtech) enabling the quick set-up of large throughput crystallisation screens in 96-well plates. The screening strategy chosen was based on the commercial sparse matrix screens (Jancarick and Kim 1991). Four variants of ADPGK could be purified in large enough quantities to allow crystallisation screening, rhADPGK, rhADPGKΔ50, rmADPGK and rmADPGKΔ51. In addition to different protein constructs, attempts were made to improve the chance of successful crystallisation by varying incubation temperature (4 ˚C and 21 ˚C), different protein concentrations (between 2 and 20 mg/ml), co-crystallisation of a ligand or substrate, reductive methylation (Walter et al. 2006; Shaw et al. 2007), limited proteolysis (Wernimont and Edwards 2009) and using different commercially available crystallisation screens (see section 2.1.7). Limited proteolysis can improve the crystallisation behaviour of a protein by producing smaller, less flexible, fragments through proteolytic digestion.

116 The reagents for limited proteolysis were purchased from Jena Bioscience (Germany) as the Floppy Choppy kit and samples prepared as described in section 2.6.2. Pre- screening of suitable conditions was performed prior to setting up the crystallisation trials. Based on outcomes of earlier crystallisation screens the concentration of rmADPGK was adjusted to 9 mg/ml and incubated with α-chymotrypsin, trypsin, subtilisin and papain, each in 1/10, 1/100 and 1/1000 dilutions of the protease. Samples were analysed by SDS-PAGE after 30 minutes incubation. As shown in Figure 56, at a dilution factor of 1/10, subtilisin and papain digested the target protein completely within 30 minutes, and were therefore deemed not suitable for this experiment. Trypsin and α-chymotrypsin were chosen as more suitable proteases since they seemed to have only limited access to cleavage sites of ADPGK, leading to fewer degradation products. Based on the results of the pre-screening these two proteases were used at a 1/200 dilution for the crystallisation screens.

Figure 56: Limited proteolysis of mouse ADPGK.

Proteolysis fragment screening of mouse ADPGK using the Floppy Choppy kit (Jena Bioscience). 1 μg

of protein was loaded per lane. The gels show samples of ADPGK incubated with α-chymotrypsin,

trypsin, subtilisin and papain, each in 1/10, 1/100 and 1/1000 dilutions of the protease (labelled accordingly). Lane B is the control reaction of ADPGK incubated with proteolysis buffer only. Lane C is untreated protein.

Another form of protein modification to improve crystallisation chances is reductive methylation. With this method, solvent accessible lysine residues and the N-terminus will be methylated (Walter et al. 2006; Shaw et al. 2007). This will affect protein solubility by altering the isoelectric point of the protein slightly. Also, the protein will become slightly more hydrophobic, which can allow for previously unavailable hydrophobic contacts to be formed upon crystallisation. Mouse ADPGK was purified by size-exclusion chromatography as described in section 2.5.10 and reductive methylation was performed as described in section 2.6.3. Native blue PAGE confirmed the methylation of the product after further purification by size-exclusion chromatography as can be seen in Figure 57.

Figure 57: Reductive methylation of mouse ADPGK.

The native blue PAGE gel shows mouse ADPGK non-methylated (1) and methylated (2). Due to the

change in the proteins pI caused by the methylation of lysines, the methylated protein migrates faster in the gel during electrophoresis.

Methylation of the surface lysine residues increases the overall negative charge of the protein at the given pH for electrophoresis. This leads to a faster migration of the methylated sample when compared to the non-methylated control sample during electrophoresis (see Figure 57). The added molecular weight of 28 Da per methylated lysine appears to be negligible in this case in decreasing migration in the gel. The methylated as well as the non-methylated mouse ADPGK proteins were used to set up

118 crystallization screens in 96-well plate format. While confirmation of the successful methylation by mass spectrometry is recommended (Walter et al. 2006; Shaw et al. 2007), this was not possible due to technical difficulties at the time of the experiment. Co-crystallisation of ADPGK and its substrates and products was attempted with D-

glucose (10 mM), D-glucose-6-phosphate (10 mM), AMP (10 mM), MgCl2 and ADP

together (6 mM each), non-hydrolysable ADP-analog AMPCP (5 mM) and D-glucose, MgCl2 and ADPGK together (5 mM each). In the case of co-crystallisation with

glucose small, round crystals were obtained in many conditions, including the control experiment without protein, which indicates that the small crystals were likely to be glucose. Also X-ray diffraction patterns collected from these crystals showed the lowest resolution spots at around 5 Å and a large spot separation, indicating crystals with unit cell dimensions too small to contain protein. None of the above conditions tested led to diffracting protein crystals.

Initial crystals of ADPGK could only be obtained for construct rmADPGKΔ51, a truncated version of mouse ADPGK, missing the first 51 N-terminal amino acids. The purification of rmADPGKΔ51 is described in section 3.2.9 above. The initial hits of needle-type crystals could be seen in a 96-well plate set up with the Mosquito robot as sitting drops with 400 nl total drop volume and a screen set up manually in 24-well plates as hanging drops with 2 μl drop volume from a protein solution at a concentration of 9 mg/ml after an incubation time of 2 weeks at 21 ˚C in condition A9 of the JCSG+ HT-96 screen (Molecular Dimensions) and condition D10 of the Structure Screen I + II HT-96 (Molecular Dimensions). Condition A9 of the JCSG+ screen contains 0.2 M NH4Cl, 20% (w/v) PEG 3350. Condition D10 of the Structure

Screen contains 0.05 M potassium dihydrogen phosphate and 20% (w/v) PEG 8000. Improved crystals of rmADPGKΔ51 were obtained from optimisation when combining the initially successful condition with the Silver Bullet Bio screen (Hampton Research) as an additive screen. Improved crystals were found in conditions A1 and H2. Crystals from both screens are shown in Figure 58. Condition A1 of the Silver Bullets Bio screen contained 0.16% (w/v) L-citrulline, 0.16% (w/v) L-ornithine hydrochloride, 0.16% (w/v) urea, 0.16% (w/v) oxalic acid, 0.16% (w/v)

kanamycin monosulfate, 0.16% (w/v) L-arginine and 0.02 M HEPES sodium pH 6.8. Condition H2 contained 0.16% (w/v) adenosine 5′-triphosphate disodium salt hydrate,

0.16% (w/v) pyridoxal 5-phosphate monohydrate, 0.16% (w/v) creatine monohydrate, 0.16% (w/v) thymine, 0.16% (w/v) L-malic acid sodium salt, 0.16% (w/v) spermine

and 0.02 M HEPES sodium pH 6.8. The optimised crystal grown in condition A1, as shown in Figure 58, was suitable for X-ray diffraction and taken to the Australian synchrotron for data collection.

Figure 58: Crystals of ADPGK.

Crystals of rmADPGKΔ51. (a) Crystals of rmADPGKΔ51 grown in condition A1 of the Silver Bullets

Bio screen. (b) Non-optimised crystals of rmADPGKΔ51 grown in condition A9 of the JCSG+ screen.

(c) Non-optimised crystals of rmADPGKΔ51 grown in condition D10 of the JCSG+ screen. (d)

Crystals of rmADPGKΔ51 grown in condition H2 of the Silver Bullets Bio screen.