Pr thermcontrol software

DSF was performed
using Thermo Fisher QuantStudio 7 Flex Real-Time PCR System. Experiments
were carried out in 384-well plates with 5 μL reaction volumes.
The assay buffer was 20 mM Tris pH 7.5, 300 mM NaCl, 0.5 mM TCEP,
and 2.4% v/v DMSO. AHR-ARNT (0.2 mg/mL, 2.22 μM) was mixed with
14-point serial dilutions of Picoberin ranging from 50 nM to 240 μM.
The Tm shifts were calculated with the Protein Thermal Shift software
from the RT-PCR instrument.
To test a potential binding of Picoberin
to ARNT alone, 0.5 mg/mL His-ARNT was incubated with 10 or 200 μM
Picoberin (1% (v/v) DMSO) for 10 min (10 μM) or 30 min (200
μM) at 22 °C in HEPES buffer (20 mM HEPES, 200 mM NaCl,
1 mM TCEP, and 5% (v/v) glycerol). The thermal protein stability from
20 to 90 °C (1 °C/min) was measured by means of the intrinsic
tryptophan/tyrosine fluorescence using the Prometheus NT.48 (NanoTemper
Technologies, DE). Melting scans, first derivatives of melting scans,
and melting temperatures were analyzed using the PR.ThermControl software
(NanoTemper Technologies, DE).
Graphs were generated with GraphPad
Prism 9.0 (GraphPad Software,
Inc., USA).

Protein unfolding was monitored at 330 nm and 350 nm using a Prometheus NT.48 (NanoTemper). FIS1 and FIS1ΔN were prepared at a final concentration of 25 μM in 100 mM Hepes, pH 7.4, 200 mM NaCl, 1 mM DTT, 0.02% NaN₃. Approximately 10 μl per sample were loaded into Prometheus NT.48 Series nanoDSF high sensitivity capillaries (NanoTemper). A melting scan was performed using the Pr.ThermControl software (NanoTemper) with an excitation power of 100%, temperature range of 25 °C to 95 °C, and temperature ramp of 1 °C/min. The midpoint of the thermal unfolding curve (T_m) was determined as the temperature corresponding to the maximum value of the first derivative of the 330 nm/350 nm fluorescence signal. Data were imported into R using readxl where box and whisker plots were generated using Tidyverse with T_m value represented on the y-axis and protein construct on the x-axis. Three biological replicates, each with three technical replicates, were used for T_m determination with error represented as SD.

The thermal stability of D1_401-560 mutants was measured using label-free, native differential scanning fluorimetry (nanoDSF; Prometheus NT.48, NanoTemper). Each mutant protein was diluted with 1X PBS until the final concentration became 0.15 mg/ml. The tryptophan residues of the proteins were excited at 280 nm, and the fluorescence intensity was recorded at 330 and 350 nm. The temperature of the measurement compartment increased from 25 to 85°C at a rate of 1°C/min. Two-state Tm₁s were obtained using PR.ThermControl Software (NanoTemper).

sE proteins were stored in 1× PBS (pH 7.4) and diluted to 2, 4, or 8 μM into the same buffer and maintained on ice before analysis. Thermal melts were performed using a NanoTemper Prometheus NT.48, in duplicate, by transferring 10 μl of diluted protein into a capillary and monitoring fluorescence at 330 or 350 nm, as designated, with a ramping rate of 1°C min⁻¹ from 15° to 95°C. Melting transition points (T_m) were calculated using Nanotemper PR.ThermControl software. For van’t Hoff analysis, thermal melts were performed at concentrations ranging from 2 to 16 μM with same settings as single concentration measurements. The concentration tested and the measured T_m¹ were used for 1/T and ln(K_d) values, respectively, as previously determined (24 (link)). Ninety-five percent confidence limits for the homodimer K_ds at 37°C were obtained using error propagation and the linear regression fits used for van’t Hoff analysis.

Protein thermostability was estimated using a label-free fluorimetric analysis in a Prometheus NT.Plex instrument (NanoTemper Technologies, Munich, Germany) as described in our previous works [20 (link),31 (link)]. A total of 37 µM of S100A1 (the concentration corresponds to the protein concentration in a calorimetric cell at the end of ITC titration) was loaded into NanoDSF grade capillaries (NanoTemper Technologies, Munich, Germany) in the presence of either 1 mM Ca²⁺, 1 mM Zn²⁺, or neither. The concentration of zinc or calcium ions varied from 18.5 to 296 µM (excesses 0.5 to 8). The capillaries were loaded onto the instrument and heated from 20 °C to 110 °C at a 1 K/min heating rate with the excitation laser power set at 100%. The unfolding transition points (T_m) were determined from the first derivative of the changes in the emission wavelengths of ratio of tryptophan fluorescence intensities at 350 nm and 330 nm (I₃₅₀/I₃₃₀), which were automatically identified by the pre-installed PR.ThermControl software (NanoTemper Technologies, Munich, Germany). In addition, the aggregation temperatures (T_agg) were determined based on first derivative of temperature dependence of light scattering at 350 nm, using the same equipment.

Thermal unfolding analyses were performed with dye-free differential scanning fluorimetry (nanoDSF), which monitors the changes in the spectral properties of tryptophan and tyrosine residues due to changes in their environment caused by unfolding (Alexander et al., 2014 (link)). The proteins have the following number of tryptophans: hOGC (two); hDIC (one); bOGC (five); bGIC-1 (five); bGIC-2 (four). Approximately 0.6 μg of protein and 10 mM substrate were added into a final volume of 10 μl purification buffer B, and the samples were loaded into nanoDSF-grade standard glass capillaries. The temperature was increased by 4°C every minute from 25 to 95°C, the intrinsic fluorescence was measured in a Prometheus NT.48 nanoDSF device, and the apparent melting temperature (Tm) was calculated with the PR.ThermControl software (NanoTemper Technologies).