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.
3.6.2 In silico protein modelling
To obtain tertiary structures of proteins of interest, in silico models had to be generated. These were obtained by using the following homology-based programs, based on the amino acid sequence (Table 34).
Table 34
Programs used for homology-based structure modelling
Usage Software
Hierarchical protein modelling I-TASSER 202,203 Markov model based modelling PHYRE v2.0 204
3.6.3 Dynamic light scattering (DLS)
To analyze the distribution of radii, present in a solution, DLS experiments were carried out using the SpectroSize™ 300 (Xtal Concepts, Hamburg, Germany). In advance protein solutions were centrifuged at 21000 xg for at least 10 min at 4 °C to precipitate large particles. Afterwards 20 µl of the measured protein were transferred to a Hellma™ Far UV quartz cuvette with a light path length of 10 mm. Scattering was measured twenty times for 20 s at 660 nm and 20 °C. The viscosity of the solution was adjusted in the program according to the components of the buffer.
3.6.4 Circular dichroism (CD) spectroscopy
To obtain insights into the secondary structure of proteins circular dichroism (CD) measurements were performed using the J-815 spectrometer (JACO International, Tokyo, Japan). Due to the interference of chloride with CD spectroscopy proteins solutions were dialyzed using an identical buffer containing fluorine as a substitute. As an alternative, solutions were diluted 1:25 with ddH2O to overcome the absorbance of chloride in the far UV spectrum 205. The instrument was calibrated according to the manufacturer’s specifications. The ellipticity of the sample was measured in a 1 mm quartz cuvette in a wavelength interval ranging from 185-260 nm. The baseline recorded for the corresponding buffer was subtracted. To compare different conditions the observed ellipticity was normalized. This molar ellipticity ([θ]) was calculated being defined as
[&] = (° × 10 × ×
where m° is the ellipticity in millidegrees, MW is the molecular weight in Da, l is the light path length in cm and c is the protein concentration in mg ml-1.
3.6.5 Small-angle X-ray scattering (SAXS)
SAXS data was collected at beamline P12 operated by EMBL Hamburg at the PETRA III storage ring (DESY, Hamburg, Germany). Scattering data was collecting using a photon counting Pilatus3 X 2M pixel detector (Dectris, Baden-Daettwil, Switzerland) with a sample-detector distance of 3.1 m. A scattering vector q (where q = 4π sinθ/λ, 2θ is the scattering angle and λ is the radiation wavelength) range from 0.03 to 4.8 nm was recorded (Table 35A).
For batch measurements, monodisperse protein solutions in form of concentration series between 1 and 10 mg ml-1 were used. Scattering of protein solutions and corresponding buffers was detected successively 20 times for 45 ms. As a first data processing step all 20 measurements were normalized to H2O and averaged. Afterwards the buffer scattering data was subtracted. All these steps were performed using the SASFLOW pipeline.
As a next step the radius of gyration (Rg) and the forward scattering intensity (I(0)) were calculated using the Guinier fit by applying AUTORG. In a next step all data points at very low angles, which were neglected for the Guinier fit, were trimmed. Using an indirect Fourier transformation, the P(r) function was calculated by DATGNOM. The maximum dimension (dmax) was defined manually and had to result in a smooth, not too elongated P(r) curve, with a reasonable fit to the experimental data. These programs were used as part of the PRIMUSQT package, which is part of the ATSAS suite.
To calculate theoretical radii of gyration (Rg) for proteins, Flory’s equation was used, which is defined as
+= x -.
where R0 is a constant, which depends on the type of protein, in Å, N is the number of amino acid residues and ν is a scaling factor 206.
To evaluate the fit, standardized residuals (Δ/σ) were plotted being defined as
∆
/= 0123( ) − 04 5( ) /(04 5( ))
where Iobs(s) is the detected scattering intensity, Iexp(s) is the expected intensity and σ(Iexp(s)) is the standard deviation of the expected intensities
In a next step the calculated radius distribution was used to generate ab initio models of the protein investigated using DAMMIF or GASBOR. Ab initio models were generated 20 times and compared using DAMAVER. The finals model was chosen based on the normalized spatial discrepancy (NSD).
To adjust the in silico models to the scattering data, different programs were applied. In case of low flexibility SREFLEX was used to refine the models. Afterwards models were evaluated by using CRYSOL. In case of higher predicted flexibility EOM was used. Therefore, the proteins of interest were divided into domains, which were predicted to be more ordered (Table S2, S3). Inter-domain regions were defined as flexible and allowed for adjustment of the model to the obtained scattering data.
Table 35
(A) SAXS data-collection parameters
Instrument PETRA III (DESY, Hamburg, Germany), Beamline P12 207 Detector Photon counting Pilatus3 X 2M pixel detector (253 x
288 mm2) (Dectris, Baden-Daettwil, Switzerland) Sample-detector-distance (m) 3.1
Wavelength (nm) 0.124
Focal spot (mm) 0.2 x 0.12
Table 35 (A) continued
s measurement range (nm-1) 0.03-4.8
Exposure time (ms) 45
s-axis calibration Silver behenate Sample temperature (°C) 20
(B) Software used for SAXS data processing
Usage Software
Primary data reduction and processing
SASFLOW v3 208
Program suite ATSAS v2.8.3 209,210
Graphical User Interface PRIMUSQT v2.8.3 211 Calculating the Guinier fit AUTORG v2.8.3 209,210 Calculating the P(r) function DATGNOM v2.8.3 209,210 Generating ab initio model (dummy
atom model)
DAMMIF 212 Generating ab initio model (chain-like
dummy residues)
GASBOR 213,214 Aligning and evaluation of ab initio
models DAMAVER 215
Fit improvement of atomic models
against scattering data SREFLEX 216 Fit improvement of flexible proteins
against scattering data EOM v2.0 217,218 Evaluation of theoretical scattering
curves of atomic models
CRYSOL 219 Flexible refinement of atomic models SASREF 214 Generation of form factor files FFMAKER 211 Mixture analysis of polydisperse systems OLIGOMER 211
Prior to all measurements different concentrations of bovine serum albumin (BSA) were measured to test for technical errors. In addition, obtained values were used to calculate theoretical MW-values for the proteins of interest. The theoretical MW being defined as
$= 6
0(0)6× 0(0)$
where MWS is the molecular weight of BSA in Da, I(0)S is the forwards scattering intensity of BSA and I(0)I is the forwards scattering intensity of the protein of interest 220.
3.6.6 Inline size-exclusion chromatography SAXS (SEC-SAXS)
Inline SEC-SAXS was performed using a Superose™ 6 Increase 10/300 GL (GE Healthcare, Chicago, United States) column with a flow rate of 0.5 ml min-1. Continuous 1 s data-frame measurements of 50 µl of sample with 120 frames ml-1 were performed (Table 36). Data was pre-processed using CHROMIXS 221. 25 frames from each sample were selected manually. 50 corresponding buffer
frames were selected automatically. Afterwards scattering data was processed as described in 3.6.5.
Table 36
SEC-SAXS parameters
HPLC 1260 Infinity II Bio-Inert sytem (Agilent, Santa Clara, United States)
SEC column Superose™ 6 Increase 10/300 GL (GE Healthcare, Chicago, United States)
Injection volume (µl) 50 Flow rate (ml min-1) 0.5 Frame rate (frames ml-1) 120
Exposure time Continuous1 s data-frame measurements (B) Software used for SEC-SAXS data processing
Usage Software
Data frame selection CHROMIXS v2.8.3 221
3.6.7 Electrostatic potential analysis
To calculate the electrostatic potential of different structures, the APBS-PDB2PQR suite was used.
In a first step the pdb files, which were analyzed, were prepared using PDB2PQR to account for missing information, which is needed to solve the equations of continuum electrostatics.
Therefore, PDB2PQR was used with developer-recommended settings and a pH of 7. In a second step the obtained results were used to calculate the electrostatic potential of the solvent accessible surface. All calculations were performed using the web-based versions of the programs. Results were visualized using PyMOL.
Table 37
Software used for the calculations of electrostatic potentials of proteins
Usage Software
File preparation PDB2PQR v2.1.1 222 Electrostatic potential
calculation APBS v1.5 223
3.6.8 Protein structure visualization
All structures were visualized using PyMOL. Ab initio models were depicted as spheres with a radius corresponding to the calculated one obtained from DAMMIF or GASBOR. Refined in silico structures were depicted in the cartoon style with flexible areas being visualized in the surface style. Proteins or domains were aligned using the align function of PyMOL. The solvent accessible surface was as well depicted in the surface style. Electrostatic potentials were illustrated using the APBS Tools v2.1 plugin.
Table 38
Software used for protein structure visualization
Usage Software
Visualization PyMOL v2.1.1 224 Electrostatic potential
visualization APBS Tools v2.1 223