Full length 6xHis-TF-AbWSD1 fusion protein was used for crystallisation because it was not possible to separate 6xHis-TF and AbWSD1 after protease-mediated cleavage. TF is a member of the group of ribosome associated chaperones (Kramer et al., 2002) and has a physiological role in assisting nascent peptides in correct folding (Deuerling et al., 1999). Thus, its presence during crystallisation might have a beneficial effect on crystallisation, which may even compensate disadvantages associated with the flexible domain architecture. Hydrophobic patches of AbWSD1 could be shielded from interaction with hydrophobic parts of other proteins by the TF, preventing the formation of aggregates and possibly promoting crystal formation (Figure 27 B).
All crystallisation experiments were done with the assistance of Dr. Karin Kühnel. Most of the crystallisation screens were pipetted by Felix Lambrecht. The screening process was started with commercially available screens in a 96-well format (Table 5) using a Cartesian pipetting robot. In all
conditions containing Ca2+-ions, spherulites were formed (Figure 29 A). Whether those spherulites were protein or derived from precipitation of calcium glycinate, originating from the glycine buffer, remains elusive. In addition, needle or snowflake-like structures were formed in numerous conditions after an average of approximately one week (representatively shown in Figure 29 B).
Almost all of these conditions contained 0.2 M MgCl2. Moreover, isopropanol, 1,6-hexandiol, ethanol, tert-butanol, 2-methyl-2,4-pentadiole (MPD) as well as different Jeffamines were promising precipitants in many cases. Except for the Jeffamines, which are polyetheramines, all of these compounds are alcohols. Since alcohols are substrates of AbWSD1, it is conceivable that the enzyme could be stabilised by these precipitants. The crystalline structures found in the commercial screens did not grow bigger and were too small for further analyses. Hence, grid screens (listed in 3.6) were designed to improve crystal formation by systematically varying pH values and the precipitant concentrations. In the case of ethanol, two crystals grew, which both had a size of at least 100 µm in each dimension (Figure 29 C&D). Both crystals were formed in conditions containing 17.3 % (v/v) of ethanol, 0.2 M MgCl2 as well as trypsin in a protease/protein ratio of 1:200 (w/w) in a sitting drop 96-well plate. Both drops were pipetted with 200 nl protein solution (20 mg/ml in 20 mM glycine/NaOH pH = 10, 150 mM NaCl) and 100 nl of precipitant. The buffers in the two conditions were 0.1 M HEPES pH = 6.5 (Figure 29 C) and 0.1 M Tris/HCl pH = 8.0 (Figure 29 D), respectively. Crystals were first detected in images acquired after 56 days of incubation at 4 °C and were reproducibly obtained under the mentioned conditions thereafter. Crystals were transferred into a cryoprotectant composed of precipitant supplemented with 30 % ethylene glycol to prevent ice formation and flash-cooled in liquid nitrogen. Diffraction data were collected by Dr. Karin Kühnel at beamline X10SA at the Swiss Light Source in Villigen, Switzerland. The crystal grown in 0.1 M Tris/HCl pH = 8.0, 0.2 M MgCl2, 17.3 % EtOH, trypsin, 1:200 (w/w) (Figure 29 D) diffracted to a resolution of up to 2.1 Å (Figure 30). The crystal belonged to the centred tetragonal space group I442, with unit cell dimensions of a = b = 117.1 Å, c = 141.2 Å and α = ß = γ = 90 °. Statistics of the collected dataset are shown in Table 18. The crystal which grew in a similar condition with 0.1 M HEPES pH = 6.5 did not diffract, probably due to damage in the freezing process.
The unit cell content can be analysed by calculation of the Matthews Coefficient (VM). This gives an estimation of the solvent content and the number of molecules present in the asymmetric unit. The asymmetric unit is the smallest fraction of the unit cell and by applying of the symmetry operations, the entire unit cell is assembled from an asymmetric unit. In space group I422, the unit cell is composed of 16 asymmetric units. VM is the ratio of the unit cell volume divided through the molecular weight of the protein multiplied with the number of asymmetric units and the number of molecules in the asymmetric unit. Based on the calculated Matthews Coefficient (VM), it was concluded that a crystallisation of the complete 6xHis-TF-AbWSD1 fusion protein in the diffracting crystal was very unlikely. Under the measured conditions, VM for a single molecule with a molecular weight of 106 kDa per asymmetric unit is 1.14. This VM corresponds to a solvent content of merely 7.74 %, a value too low to represent a realistic solvent content of a protein crystal. Typically, protein crystals have a solvent content in the range of 25-80 %.
RESULTS
Figure 29: Crystallisation of the 6xHis-TF-AbWSD1 fusion protein. A) In all conditions containing Ca2+, spheroblasts were formed (indicated by black arrows). B) Example of small, spider-like crystals (indicated by black arrows). Crystals shown in C) (0.1 M HEPES pH = 6.5, 0.2 M MgCl2, 17.3 % EtOH, trypsin, 1:200 (w/w)) and D) (0.1 M Tris/HCl pH = 8.0, 0.2 M MgCl2, 17.3 % EtOH, trypsin, 1:200 (w/w)) were tested for diffraction at the Paul Scherrer Institute in Villigen, Switzerland. The crystal shown in D) diffracted to a resolution of 2.1 Å.
Under the given parameters, VM for a protein of 52000 Da is 2.33, which corresponds to a calculated solvent content of 47.2 %. Hence, it is very likely that the asymmetric unit of the crystal contains either a single AbWSD1 molecule or a single TF molecule, since both proteins are similar in size. In order to determine the structure of the crystallised protein, molecular replacement using the Vibrio cholera TF structure (PDB accession code 1T11) (Ludlam et al., 2004) and the E. coli TF structure (PDB accession code 1W26) (Ferbitz et al., 2004) was tried, but attempts were not successful. Moreover, molecular replacement was tried with the surfactin synthetase subunit 3 (PDB accession code 2VSQ) (Tanovic et al., 2008), the structure which was chosen by Phyre (Kelley and Sternberg, 2009) for modelling of the AbWSD1 sequence. However, molecular replacement based on this structure was not successful either. Soaking of the crystals with iodide for single wavelength anomalous diffraction (SAD) phasing resulted in dissolving of the crystals. The goal was then to determine structure through SAD phasing with selenomethionine labelled protein. The protein was expressed in minimal medium supplemented with selenomethionine. Purification of 6xHis-TF-AbWSD1 carrying selenomethionine under the same conditions as described for the unlabelled 6xHis-TF-AbWSD1 produced sufficient amounts of protein for further crystallisation screenings (data not shown). However, respective crystallisation screenings were ongoing at the time of writing this thesis, but hadn’t been finished.
Figure 30: X-ray diffraction pattern of 6xHis-TF-AbWSD1. The respective protein crystal of 6xHis-TF-AbWSD1 was grown in 0.1 M Tris/HCl pH = 8.0, 0.2 M MgCl2, 17.3 % EtOH, trypsin, 1:200 (w/w).
Table 18: Statistics of the X-ray diffraction data set which was collected for the 6xHis-TF-AbWSD1 protein crystal.
parameter value
Beam line SLS X10SA
Detector distance (mm) 270
ϕstart/ oscillation Δ ϕ (°) 259° / 0.5°
Exposure time (s) 0,5 s
Beam intensity 40 %
λ (Å) 0.979 Å
No. of frames 180
Space group I422
Cell dimensions
a, b, c (Å) 117.1, 117.1, 141.2
α, β, γ (°) 90, 90, 90
Resolution (highest res. shell) (Å) 50-2.10 (2.22-2.10)
Rmerge (%) 7.7 (60.2)
no. of observed reflections / unique reflections 381836/29155 (58900/4565)
I/σ (I) 26.6 (4.3)
RESULTS