3.5.1 Protein sequences
Amino acid sequences for the investigated recombinant proteins are listed in the supplement (Table S1). Due to the removal of the GST tag using the PreScission™ protease eight additional amino acids are located at the N-terminus of each protein. Therefore, each amino acid position mentioned in this thesis does not correspond to the TAIR reference sequence database 162,163, but it is shifted by eight amino acids.
3.5.2 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
To analyze proteins in general, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. Therefore, acrylamide gels were cast using the Mini-Protean® Tetra Handcast System (Bio-Rad, Hercules, United States). Spacer plates with 0.75 mm integrated spacers were used. All gels were run using a constant 40 mA per gel for at least 45 min. All gels were cast after the following recipes (Table 30).
Table 30
(A) SDS-PAGE stacking gel composition
Ingredient Concentration
Tris-HCl pH 6.8 130 mM
Acryl/bisacrylamide mix
(37.5:1) 5.1 %
SDS 0.1 %
APS 0.1 %
TEMED 0.001 %
(B) SDS-PAGE seperating gel composition
Concentration
Ingredient 10 % 12 % 15 %
Tris-HCl pH 8.8 382 mM 382 mM 382 mM
Acryl/bisacrylamide mix (37.5:1) 10 % 12 % 15 %
SDS 0.1 % 0.1 % 0.1 %
APS 0.1 % 0.1 % 0.1 %
TEMED 0.001 % 0.001 % 0.001 %
3.5.3 Colloidal Coomassie G-250 staining
To detect proteins after SDS-PAGE, gels were stained using colloidal Coomassie G-250 staining 196. Therefore, gels were washed three times for at least 10 min using ddH2O. Afterwards gels were stained for at least 1 h. I necessary, gels were destained using destain or ddH2O. Gels were stored in ddH2O. Colloidal Coomassie G-250 stain and destain were prepared after the following recipe (Table 31) and protocol 196.
Table 31
Colloidal Coomassie G-250 stain and destain recipe
Ingredient Concentration
Stain
Coomassie Brilliant Blue G-250 0.02 % Aluminium
sulfate-(14-18)-hydrate 5 %
Ethanol 10 %
Phosphoric acid 2 %
Table 31 continued
Ingredient Concentration
Destain
Ethanol 10 %
Phosphoric acid 2 %
3.5.4 Western blot analysis
To detect specific proteins after gel electrophoresis by SDS-PAGE (see chapter 3.5.2) semi-dry Western blotting was performed. Therefore, unstained SDS-gels were washed three times for 5 min with H2O and equilibrated in transfer buffer. Roti®-NC nitrocellulose membrane (Carl Roth, Karlsruhe, Germany) and Whatman™ 3MM Chr chromatography paper (GE Healthcare, Chicago, United States) were as well equilibrated using transfer buffer. For the semi-dry Western blot the FastBlot B43 (Biometra, Göttingen, Germany) was used with a sandwich containing two Whatman™ papers, the nitrocellulose membrane, the SDS-gel and again two Whatman™ paper. All air bubbles were removed and the transfer was performed using 60 mA per gel for 65 min.
In a next step the nitrocellulose membrane was washed for 5 min using TBS-T buffer and incubated overnight in blocking solution at 4 °C and continuous shaking. Afterwards the nitrocellulose membrane was washed twice for 5 min using TBS-T buffer and the primary antibody Anti-Glutathione-S-Transferase IgG (Sigma-Aldrich, St. Louis, United States) was added in a dilution of 1:5000 in 10 ml blocking solution and incubated at RT for 2 h an continuous shaking. After that the primary antibody was discarded and the nitrocellulose membrane was washed three times for 5 min using TBS-T buffer. The secondary antibody Anti-Rabbit IgG HRP-conjugate (Merck, Darmstadt, Germany) was then added in a dilution of 1:5000 in 10 ml blocking solution and incubated as well for 2 h at RT and continuous shaking. The secondary antibody was discarded as well and the nitrocellulose membrane washed three times for 5 min using TBS-T buffer. The detection of the luminescence signal was performed by incubating the nitrocellulose membrane with Clarity™ Western ECL Substrate (Bio-Rad, Hercules, United States) and using the ChemiDoc™ Touch Imaging System (Bio-Rad, Hercules, United States).
Table 32
Buffers used for Western blot
Buffer Components
Transfer 50 mM Tris-HCl pH 8.3, 40 mM glycin, 20 % methanol
TBS-T 50 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.05 % Tween™ 20
Blocking solution 50 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.05 % Tween™ 20, 5 % skim milk powder
3.5.5 Protein concentration determination
All protein concentrations were determined measuring the absorbance at 280 nm. Each sample was normalized to the corresponding buffer. To correct for differences in tryptophane and tyrosine content, the corresponding extinction coefficients and the related molecular weight were considered for the calculations. The concentrations (c) in mg ml-1 were calculated according to the Beer-Lambert law as
= × ×
where Abs280 nm is the absorption at 280 nm in AU, ε is the extinction coefficient in M-1 cm-1, l is the light path length in cm and MW is the molecular weight in Da.
3.5.6 Microscale thermophoresis (MST)
To investigate protein interactions and determine the binding affinity MST experiments were carried out. Therefore, proteins of interest were labelled using the Monolith NT™ Protein Labelling Kit Green (NanoTemper Technologies, Munich, Germany) according to manufacturer’s specifications. Measurements were performed using the Monolith NT.115 (NanoTemper Technologies, Munich, Germany).
3.5.7 Mass spectrometry (MS)
To identify and analyze singular protein bands from SDS-PAGE experiments, mass spectrometry (MS) measurements were performed. These were carried out by the Molecular Plant Genomics research unit of Julia Kehr (Institute of Plant Science and Microbiology, Hamburg, Germany). All experiments were conducted and evaluated by either Patrizia Hanhart, Anna Ostendorp or Julia Kehr.
3.5.8 Native MS
To analyze the interactions between two proteins under native conditions, native MS experiments were performed. These were planned and carried out in corporation with the Dynamics of Viral Structures research group of Charlotte Uetrecht (Heinrich Pette Institute, Hamburg, Germany). All experiments were conducted and evaluated by Julia Lockhauserbäumer.
For native MS proteins were used at concentrations of 5–11 µM. Purified proteins were buffer exchanged prior to MS analysis to 250 mM ammonium acetate at pH 9.0 or 7.5, via Vivaspin® 500 Centrifugal Concentrators (Sartorius, Göttingen, Germany) with a MWCO of 10 kDa at 15,000 × g.
Native mass spectra were measured at 25 °C on a Q-ToF instrument (Waters, Milford, United States) modified for high mass experiments with a nano-electrospray ionization (ESI) source in positive ion mode 197. Samples were directly infused from gold-coated electrospray capillaries without any accessory chromatographic separation. The voltages and pressures were optimized for noncovalent protein complexes 198.
The gas pressures were set to 10 mbar in the source region and 1.8 × 10-2 mbar argon in the collision cell. Mass spectra were recorded with applied voltages for the capillary, cone and collision cell of 1.25–1.35 kV, 150 V and 5–150 V, respectively, optimized for minimal complex dissociation. For the calibration of the raw data MassLynx (Waters, Milford, United States) and a
25 mg ml-1 cesium iodide spectrum from the same day was used. MassLynx was used to assign peak series to protein species and to determine the mass after minimal smoothing.
3.5.9 Thermofluor assay
To improve protein stability, the effects of different buffers on the protein of interest were compared. Therefore, a thermofluor assay was performed to determine the melting temperature (Tm) of the protein of interest. Thus, to the protein in its original buffer different buffers and SYPRO™ Orange Protein Gel stain (Thermo Fisher Scientific, Waltham, United States) were added.
Afterwards a melting curve was recorded with the detected fluorescence corresponding to the amount of the protein being unfolded.
The analysis was performed after Boivin et al. 199, using the suggested buffers for global parameters. Measurements were performed using a QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific, Waltham, United States). To calculate the melting temperature Tm of the protein of interest under different buffer conditions, the fluorescence of the melting curve was fitted against a modified Boltzmann equation 200, for which the relative fluorescence units (RFU) are defined as
= + ( − )
(1 + )
where RFU is the fluorescence in arbitrary units, RFUmin and RFUmax are the minimal and maximal fluorescence at low and high temperatures, Tm is the melting temperature of the protein in °C, x is the temperature in °C and m is the slope of the curve in °C-1.
Fits were performed using OriginPro 2018G (OriginLab Corporation, Northampton, United States) with the modified Boltzmann equation. To compare the effect of different buffer environments, the thermal shift (ΔTm) was calculated being defined as
∆! = ! ("#$)− !
where Tm(POI) is the melting temperature of the protein of interest in its original buffer.