Nano-DSF measurements were performed using a Prometheus NT.48 instrument (NanoTemper Technologies, Germany). In total, 6 samples for both full-length and truncated LR were made, having pH 6.0, 7.0, or 8.0 and DDM concentration of 0.05% and 1%. First, samples with purified LR at a concentration of 1 mg ml−1 were dialyzed against 200 mM NaCl, 0.05% DDM buffer for 18 h at 4 °С. After that, the protein was introduced into 50 mM Na2HPO4/NaH2PO4, 200 mM NaCl buffer with the desired pH, and incubated at 4 °C for another 18 h. About 10 μL of each sample was loaded into UV capillaries (NanoTemper Technologies). The temperature gradient was set at 1 °C min−1 in a range from 15 to 98 °C. Protein unfolding was observed by following the change in tryptophan fluorescence at emission wavelengths of 330 and 350 nm. The ratio between the emission intensities at 350 and 330 nm (F350/F330) was used to track the structural changes with increasing temperature. Melting-point temperatures (Tm) were calculated using the peaks in the first derivative of the signal data.
Uv capillaries
Manufactured by NanoTemper
Sourced in Germany
UV capillaries are a type of laboratory equipment used to measure the absorption of ultraviolet (UV) light by samples. They are designed to hold small volumes of liquid samples and allow the UV light to pass through, enabling the measurement of the sample's UV absorbance.
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9 protocols using uv capillaries
1
Nano-DSF Analysis of Ligand Receptor Unfolding
2
Thermal Unfolding of Proteins
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For each condition, 10 μL of sample per capillary were prepared. The protein samples were loaded into UV capillaries (NanoTemper Technologies, Germany) and experiments were carried out using the NanoTemper Tycho NT.6 (NanoTemper Technologies, Germany). The temperature gradient was set to an increase of 20 °C min−1 in a range from 35 °C to 95 °C. Protein unfolding was measured by detecting the temperature-dependent change in tryptophan fluorescence at emission wavelengths of 330 and 350 nm. Inflection temperatures (Ti) were determined by detecting the maximum of the first derivative of the fluorescence ratios (F350/F330). For this, an 8th order polynomial fit was calculated for the transition region. Next, the first derivative of the fit was formed and the peak position (at Ti) was determined.
3
Thermal Unfolding of A2A-StaR2-βRIL562
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A2A-StaR2-bRIL562 purified in DM in the presence of 500 µM theophylline was used for thermal unfolding experiments. The protein was diluted in 40 mM Tris pH 7.4, 200 mM NaCl, 0.15% DM to a final concentration of 0.2 mg/ml. Following heavy dilution (~70-fold) of the protein in a buffer without ligand, the sample was considered to be in an apo-like state. Samples were supplemented with the respective ligands to a final concentration of 50 µM, with a final DMSO concentration of 5% (v/v). The control sample was supplemented with DMSO to a final concentration of 5% (v/v). Samples were incubated 30 minutes on ice before being loaded into UV capillaries (NanoTemper Technologies) and experiments were carried out using the Prometheus NT.48. The temperature gradient was set to an +1 °C/min from 20 °C to 90 °C. Protein unfolding was measured by detecting the temperature-dependent change in tryptophan fluorescence at emission wavelengths of 330 and 350 nm. The experiment was repeated four times and data analysed with the one-way analysis of variance (ANOVA) with Dunnett’s post-test. Tm values obtained for the three ligands are statistically different from the control sample with p < 0.001.
4
Nano-DSF Thermal Stability Analysis
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Nano-DSF (differential scanning fluorimetry) measurements were performed using a Prometheus NT.48 instrument (NanoTemper Technologies GmbH, Germany). Experiments on OTULINWT and OTULINL272P were carried out at 0.11 mg/mL and 0.38 mg/mL (not shown). Samples were dialysed into PBS before measurement and 10 μL of each sample were loaded in UV capillaries (NanoTemper Technologies). Temperature gradient was set at 2.5°C/min in a range from 20 to 90°C. Protein unfolding was measured by detection of change in tryptophan fluorescence at emission wavelengths of 330 and 350 nm, dependent on temperature gradient. Tm’s were calculated according to the manufacturer’s instructions. Results were confirmed by differential scanning calorimetry and CD thermal melt experiments (data not shown).
5
Nano-DSF Analysis of CyaY Thermal Stability
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Nano-DSF measurements were performed using a Prometheus NT.48 instrument (NanoTemper Technologies, GmbH, Germany). The measurements were done for CyaY without added iron and with the same Fe2+-to-CyaY ratios and buffer used in the SAXS experiments (1:2, 1:1, 2:1, 4:1, 7:1, and 10:1). The iron-incubated samples had a protein concentration of 1 mg/ml and were prepared in the same way as for the SAXS experiments. Ten μL of each sample was loaded into UV capillaries (NanoTemper Technologies). The temperature gradient was set at 0.5°C/min in a range from 20 to 95°C. Protein unfolding was detected by following the change in tryptophan fluorescence at emission wavelengths of 330 and 350 nm. The ratio between the emission intensities at 350 nm and 330 nm (F350/F330) was used to track the structural changes with increasing temperature. Data analysis was performed using the manufacturer’s software, where Tm was calculated using the peaks in the first derivative curve.
6
Determining Protein Melting Points via nanoDSF
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Nanoscale differential scanning fluorimetry (nanoDSF) measurements were performed using Prometheus NT.48 nanoDSF instrument, which monitors the shift of intrinsic tryptophan fluorescence of proteins upon unfolding by detecting the fluorescence at emission wavelengths of 330 and 350 nm. For determination of the protein melting point (Tm), the maximum of the first derivative of the fluorescence ratios F350/F330 was used.
PcPAL variants were diluted with 50 mM Tris.HCl, 100 mM NaCl pH 8.8 buffer to a final concentration of 1 mg/mL. 10 μL of each sample was loaded into UV capillaries (NanoTemper Technologies, München, Germany) and protein unfolding was detected during heating in a linear thermal ramp of 1 °C/min between 20 and 95 °C, with an excitation power of 20%. Data analysis was performed using NT Melting Control software and melting temperature (Tm) was determined by fitting the experimental data using a polynomial function, in which the maximum slope was indicated by the peak of its first derivative (F350/F330). All measurements were performed in triplicate.
PcPAL variants were diluted with 50 mM Tris.HCl, 100 mM NaCl pH 8.8 buffer to a final concentration of 1 mg/mL. 10 μL of each sample was loaded into UV capillaries (NanoTemper Technologies, München, Germany) and protein unfolding was detected during heating in a linear thermal ramp of 1 °C/min between 20 and 95 °C, with an excitation power of 20%. Data analysis was performed using NT Melting Control software and melting temperature (Tm) was determined by fitting the experimental data using a polynomial function, in which the maximum slope was indicated by the peak of its first derivative (F350/F330). All measurements were performed in triplicate.
7
Characterization of XdpB Oligomeric State
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The oligomeric state was assayed on a Yarra SEC3000 3u column (Phenomenex, HPLC) and Superdex 75 16 600 columns. The aggregation of XdpB was determined by Dynamic Light Scattering (Zetasizer 90, Malvern) between 5 to 40°C with the protein solution (0.3 mg.ml-1 to 20 mg.ml-1). The circular dichroism (CD) spectra were measured by the Chirascan-plus spectrometer (Applied Photophysics) with wavelength steps of 1 nm (range of 185–260 nm) at 20 °C and a protein concentration of 0.2 mg.ml-1 [28 ]. The buffer subtracted spectrum was analyzed by the CDNN program. Temperature induced protein unfolding was measured by detecting the change of the ratio of tryptophan fluorescence at emission wavelengths of 330 and 350 nm between 20 °C and 95 °C (step 1 °C/min) using Prometheus NT.48 with UV capillaries (NanoTemper Technologies) loaded by 10 μl of protein sample per capillary; the FMN-free XdpB wild type had a concentration of 3.3 mg/ml, and the flavinated form 2 mg/ml.
8
Thermal Unfolding of Proteins using Tryptophan Fluorescence
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For thermal unfolding experiments,
the protein solution was diluted to a final concentration of 10 μM.
For each condition, 10 μL of sample per capillary was prepared.
The samples were loaded into UV capillaries (NanoTemper Technologies),
and experiments were carried out using Prometheus NT.48. The temperature
gradient was set to an increase of 1 °C min–1 in a range from 20 to 100 °C. Protein unfolding was measured
by detecting the temperature-dependent change in tryptophan fluorescence
at emission wavelengths of 330 and 350 nm in the presence of different
concentrations of EG. For analysis, melting temperatures were determined
by detecting the maximum of the first derivative of the observed fluorescence
ratios (F350/F330). For this, the 8th order polynomial fit was performed for the transition
region. Next, the first derivative of the fit was formed and the temperature
at the peak, which is that Tm was determined.
the protein solution was diluted to a final concentration of 10 μM.
For each condition, 10 μL of sample per capillary was prepared.
The samples were loaded into UV capillaries (NanoTemper Technologies),
and experiments were carried out using Prometheus NT.48. The temperature
gradient was set to an increase of 1 °C min–1 in a range from 20 to 100 °C. Protein unfolding was measured
by detecting the temperature-dependent change in tryptophan fluorescence
at emission wavelengths of 330 and 350 nm in the presence of different
concentrations of EG. For analysis, melting temperatures were determined
by detecting the maximum of the first derivative of the observed fluorescence
ratios (F350/F330). For this, the 8th order polynomial fit was performed for the transition
region. Next, the first derivative of the fit was formed and the temperature
at the peak, which is that Tm was determined.
9
Thermal Stability of LysB-His6 Enzymes
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Stability of LysB-His6 enzymes was determined using Nano-Differential Scanning Fluorimetry (DSF) on a Prometheus NT.48 instrument (NanoTemper Technologies, GmbH, Germany). Ten microliter samples of 2 μM LysB-His6 enzymes in 50 mM Tris-HCl buffer (pH 8) were loaded into UV capillaries (NanoTemper Technologies), and subjected to increasing temperature from 20 to 95 °C with a temperature gradient of 1 °C/min. Protein unfolding was detected by tracking changes in the intrinsic tryptophan/tyrosine fluorescence (350 nm/330 nm fluorescence ratio) as a function of temperature increase. Data was analyzed using the ThermControl software V 2.0.4 (NanoTemper Technologies) and melting temperature (Tm) was calculated.
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