2.7.2 Size exclusion chromatography (SEC)The IEX step was not sufficient to remove all impurities, especially proteins with similar chemical nature, which led to the usage of size exclusion chromatography which separates proteins according to their hydrodynamic radius.The protein material used in SEC consisted of fractions collected during IEX. To prepare the protein samples for SEC, the IEX fractions containing high amounts of AaFd4 were pooled and concentrated with a centrifugal filter concentrator. A prepacked Superdex 75 column was used for preparative SEC at room temperature. To achieve a good level of separation, the protein loading volume was set to a maximum of 2 mL. Eluates were collected in 2 mL fractions with a flow rate of 1 mL/min of S75 running buffer (Table A3) and were analyzed by SDS-PAGE. Fractions corresponding to a single peak on the chromatogram and displaying high amount of AaFd4 were pooled and then concentrated by an ultrafiltration system.2.8 SDS polyacrylamide gel electrophoresis (SDS-PAGE) Discontinuous SDS-PAGE as described by Laemmli 10 was used to separate proteins according to their molecular weight and therefore to confirm the success of protein expression or to estimate the purity of proteins preparations.Protein samples were mixed with 10 ?l of 6x SDS sample loading buffer and incubated for 10 minutes at 95°C, which destroys structural elements. The samples were loaded onto a 10% SDS polyacrylamide separating gel with a 5% stacking gel and run at 25 mA. After separation, the proteins were visualized by staining the gel with a solution of Coomassie Brilliant Blue and destained until protein bands were clearly visible and the background was completely removed.The proteins were identified by molecular mass comparison with a standard protein ladder ranging from 14.4 to 116 kDa. Hardened gels were either stored in moist paper in a plastic bag at 4 °C or immediately used for gel electrophoresis.2.9 Determination of protein concentration Protein concentration was measured spectrophotometrically by the Bradford assay 11. For this purpose 20 µL of the appropriately diluted protein solution was mixed with a commercially available Bradford reagent up to a final volume of 1000 µL and incubated for 10 min at room temperature before measuring the absorbance at 595 nm. A standard calibration curve, prepared by bovine serum albumin in a range of 0-1 mg/mL, was used to estimate the concentration of AaFd4.2.10 UV-Vis spectroscopySpectra of purified AaFd4 at different redox states were recorded in an Ocean Optics spectral photometer at a spectral range of 200-700 nm.The AaFd4 concentration was set to 1.23 mg/mL by dilution with SEC buffer in order to fully exploit the linear range of the spectrometer.First an oxidized spectrum was measured, then 5 mM of a 0.5 M sodium dithionite solution was added to the sample and mixed immediately. After the reaction was finished, a spectrum of now fully reduced AaFd4 was recorded.In a second experimental series, again starting with oxidized AaFd4, an increasing amount of dithionite was added in steps of 100 µM until the reduced form was reached. For each step a spectrum was recorded for further analysis, in order to obtain a titration-like curve.2.11 Thermal shift assay (TSA)The thermal shift assay is a fast, fluorescence- and temperature-based experiment to assess the stability of proteins and to identify optimal buffer conditions for protein crystallization.For the method a small amount of protein mixed with SYPRO Orange was taken. The dye binds to hydrophobic protein regions and fluoresces. By increasing the temperature, the protein was unfolded and the increase in fluorescence emission was monitored and a melting temperature was determined.The TSA was performed using a 96-well PCR plate by measuring the corresponding values in a real time PCR machine equipped with the suitable monitoring units.Altogether, two experimental series were carried out. First, a pretest with AaFd4 and Azurin was measured to screen for suitable concentrations of protein and SYPRO Orange. Subsequently, a screening for optimal buffers with the fixed, previously found out Azurin concentration and the RUBIC Buffer Screen MD1-96 12 was done.The composition of the 96-well buffer optimization screen for azurin and the components for the experiment with varied protein concentration are listed in Table A5 and A42.12 Crystallization and data collectionCrystallization of purified and concentrated AaFd4 (~ 30 mg/mL) in 25 mM Tris/HCl + 150mM NaCl buffer at pH 8.5 was performed using the sitting drop vapor diffusion method by mixing 2 µL protein with 2 µL precipitant solution and equilibrating these against a reservoir solution. After protein and buffer administration, the plates were sealed and stored at room temperature. For the protein crystallization experiments a home-made screening kit (Table A6) with 24 different buffer conditions was used. For observing crystal formation, the plates were monitored daily with a light microscope. Diffraction data to 1.6 Å resolution for AaFd4 were collected under cryogenic conditions at 100 K in a liquid nitrogen stream at a Rigaku Micromax 007HF, a microfocus rotating anode X-ray generator producing Cu-K? radiation (1.5418 Å), on the imaging plate system mar345dtb.2.13 Phase determination and structure refinementA AaFd4 dataset, obtained from another X-Ray diffraction experiment recorded at a synchrotron beamline, was processed and scaled using iMOSFLM (indexing and integration) 13 and AIMLESS (scaling and merging) 14. The AaFd4 crystal structure was solved by molecular replacement with Phaser 15 using another ferredoxin structure (PDB ID: 1M2A) determined at 1.5 Å resolution as the search model. Positional refinement of the model was carried out using multiple rounds of reciprocal space refinement against the calculated data with REFMAC5 16 implemented in the CCP4 software suite 17 followed by manual model rebuilding and improvements through real space refinement using COOT 18.The quality of the final structure was validated by SFCHECK 19. Finally, figures were drawn using PYMOL 20 to visualize the protein structure and to show several characteristics of AaFd4.