3.5 Crystallization
3.5.1 Crystallization of ctPrp2 (270-921)
3.5.1.1 ADP bound state
Initial crystallization screening was performed with a protein concentration of 5 mg/ml and a 5× molar excess of ADP. A set of nine screens (2.1.12) was tested at 20◦Cand 4◦C. First crystals could be observed after 48 hours at 20◦C in various conditions containing dierent PEGs, salts at concentrations between 100 and 200 mM and pH values between 6 and 7.5. The initial conditions were optimized to a nal condition containing 0.1 M Tris pH 7.5, 200 mM NaCl and 15% PEG 8000 (see gure 3.23).
Figure 3.23: Protein crystals of ctPrp2 (270-921) in the pres-ence of ADP. The crystals were grown in a condition containing 0.1 M Tris pH 7.5, 200 mM NaCl and 15%
PEG 8000. The reference bar corresponds to a length of 200 µm.
The crystals were cryoprotected in reservoir solution supplied with 15% glycerol and 5% PEG 400 and ash cooled in liquid nitrogen. ctPrp2 (270-921) crystallized in the orthorombic spacegroup P212121 and diracted to a resolution of 2.1 Å. A complete dataset was measured at beamline 14.1 of BESSY, Berlin. The statistics of data collection and structure renement are shown in table 3.3. Since soaking of the crystals in KBr and AuCl3 for phasing resulted in no signicant anomalous signal, the crystallographic phase problem was solved by molecular replacement using PHASER (McCoy et al., 2007) and the structure of yeast Prp43 (PDB ID:
2XAU) as search model.
The structure was rened by iterative cycles of CNS (Brünger et al., 1998) and manual model building in Coot. The nal round of renement was performed with PHENIX (Adams et al., 2002) to overall Rwork/Rfree values of 18.74 and 22.84 % respectively (complete data statistics for renement are shown in table 3.3). The nal model of ctPrp2 (270-921) contains one molecule in the asymmetric unit and
3 Results
encompasses residues 283-921. Additional density was observed in the nucleotide binding cleft were an ADP molecule as well as a magnesium ion is present.
Table 3.3: Data collection and renement statistics of ADP bound ctPrp2 (270 - 921). Values in parentheses refer to the highest resolution shell, Ramachandran statistics were calculated with Molprobity. Resolution range (Å) 50.0 - 2.10 (2.20 - 2.10) No. of reections 150725
Resolution range (Å) 46.01 - 2.10 (2.15 - 2.10) No. of Reections 39995
For co-crystallization of ctPrp2 (270-921) with dierent nucleotides (e.g. AMPPNP, AMPPCP), co-puried ADP had to be removed from the protein. This was achieved by addition of 10 mM EDTA to the protein followed by gel ltration chromatography in a buer containing 10 mM HEPES pH 7.5, 200 mM NaCl, 2 mM, 5% (v/v) glycerol and 1 mM DTT supplemented with 10 mM EDTA. In a nal step, the EDTA was removed by a desalting column. To check whether the ADP was removed quantitatively, the nucleotide binding state was determined using a Nucleosil column (see 2.2.11.5).
By comparison with the elution prole of ADP, the chromatogram of the Nu-cleosil column shows that after treatment with EDTA, ctPrp2 (270-921) is in a nucleotide-free state ( see g. 3.24).
After a nucleotide free ctPrp2 had been puried, crystallization trials were per-formed with a protein concentration of 4 mg/ml in presence of 5× molar excess of the non-hydrolyzable ATP analogues AMPPNP and AMPPCP respectively.
Crystals could be obtained in a condition containing 0.1 M Tris pH 7.5, 200 mM
3.5 Crystallization
Figure 3.24: Nucleotide loading state of ctPrp2 after treatment with EDTA. In the chro-matogramm, absorption at 254 nm is plotted against the concentration of the eluent. As standard, an ADP sample was loaded (gray dashed line), which shows a small peak for AMP at 9.8 ml and a major peak corresponding to ADP at 13.5 ml. For the ctPrp2 sample (red line) only a very small peak can be observed at 10.3 ml.
Li2SO4 and 15% PEG 8000 . The crystals were cryoprotected in reservoir solution supplied with 15% glycerol and 5% PEG 400 and ash cooled in liquid nitrogen.
Several datasets were measured at beamline 14.1 of BESSY, Berlin. After re-nement against the already obtained structure of ctPrp2 bound to ADP it was revealed, that the protein which was crystallized in presence of AMPPNP con-tained an ADP molecule in the nucleotide binding cleft. Further analysis of the nucleotide loading state of ctPrp2 incubated with AMPPNP using a Nucleosil column showed that ADP was bound to the protein, although ADP was nei-ther present in the protein sample before incubation with AMPPNP nor in the AMPPNP solution (see Fig 3.25). It is therefore likely, that ctPrp2 (270-921) is in fact able to hydrolyze AMPPNP.
3.5.1.3 Apo state
In the rened structure obtained from datasets of ctPrp2 (270-921) crystallized in the presence of AMPPCP, no nucleotide was bound in the nucleotide binding cleft.
Instead, a sulfate ion was present at a position, which harbors the β phosphate in the ADP bound state.
The apo structure of ctPrp2 (270-921) was rened to overall Rwork/Rfree values of 21.95 and 27.29 % (complete data statistics for renement are shown in table 3.4). In the electron density map, the residues 283 - 921 could be traced, while
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6 8 10 12 14 16 18 20
0 100 200
300 ADP
AMP
AMPPNP
1
Elution volume (ml) A254(mAu)
0 20 40 60 80 100
eluent(%)
Figure 3.25: Nucleotide loading state of ctPrp2 before (red line) and after (green line) incu-bation with AMPPNP. In the chromatogram, absorption at 254 nm is plotted against the concentration of the eluent. As standard, an ADP (gray dashed line) as well as an AMPPNP (blue dashed line) sample was loaded. AMPPNP elutes at 15.2 ml, the peak corresponding to ADP is observed at 13.5 ml. After incubation of ctPrp2 with AMPPNP, a peak elutes at a volume corresponding to ADP (1, in green), indicating that AMPPNP was hydrolyzed to ADP by ctPrp2.
residues 601 - 603 located in a exible loop region are not dened. The overall shape of the protein closely resembles that of ctPrp2 (270-921) in the ADP bound state.