Pr thermcontrol

SARS-CoV-2 N, NTD and CTD thermal unfolding profiles were acquired by measuring temperature-dependent shift in their intrinsic fluorescence at 330 nm (F330) and 350 nm (F350) emission wavelengths by using a Prometheus NT.48 (NanoTemper Technologies) instrument. Purified proteins were diluted in their storage buffer to 0.3 mg mL⁻¹, 0.13 mg mL⁻¹ and 0.25 mg mL⁻¹, respectively, for the obtainment of optimal fluorescence counts, then loaded on standard capillaries (NanoTemper Technologies) and subjected to a 20–90 °C linear thermal gradient at 0.5 °C min⁻¹ rate. Inflection points of fluorescence transition corresponding to melting temperature (Tm) values were determined as the first derivative maximum of the fluorescence intensity ratio at the measured wavelengths (F330/F350). For interaction between Suramin and the proteins, the Tm shift was assessed after addition to samples of 100  $\mu$ M Suramin (Sigma-Aldrich) water solution and incubation for 1 hour at 25 °C. Suramin affinity to SARS-CoV-2 N NTD and CTD was assessed by titration of the compound over a 0.5–2000  $\mu$ M concentration range against a fixed protein amount. For each experiment, data from at least three independent measurements were processed using the PR. ThermControl (NanoTemper Technologies) software.

Thermal stability assays were performed by nanodifferential scanning fluorimetry (nanoDSF) using a Prometheus NT.48 instrument (NanoTemper Technologies, Germany). The Ca²⁺-loaded protein samples (0.2 mg/ml) were diluted in 5 mM Tris-HCl (pH 8.0), 50 mM NaCl, and 1.5 mM CaCl₂ and loaded into nanoDSF grade standard capillaries (NanoTemper Technologies, Germany). The measurements were conducted from 20 to 95 °C (with a temperature ramp of 2 °C/min) under constant monitoring of tryptophan fluorescence at 350 and 330 nm. The melting temperature (Tm) values, corresponding to the inflection points of the unfolding curve, were determined by using a PR.ThermControl (NanoTemper Technologies, Germany).

Proteins mixed to a final concentration of 30 µM in presence of 200 µM PIP₂ and incubated overnight at 4 °C were used to fill two standard grade NanoDSF capillaries (Nanotemper) and loaded into a Prometheus NT.48 device (Nanotemper) controlled by PR.ThermControl (version 2.1.2). Excitation power was pre-adjusted to get fluorescence readings between 2000 and 20000 RFU for fluorescence at 330 and 350 nm (F330 and F350, respectively), and samples heated from 20 °C to 90 °C with a slope of 1 °C/min. An apparent Tm was calculated from the inflection points of the fluorescence curves (Ratio F350/F330) for ANTH and ENTH monomeric samples, where ΔT_m = T_m mutant − T_m wild-type. To ensure we did not see any oligomerization interference effect potentially caused by domain instability, we discarded all mutants with a ΔT_m larger than 2 °C in our complex assembly study.
The scattering signal recorded (backscattering mode) was used as a stability reporter for the AENTH samples, where the mid aggregation point, T_agg, corresponds to the inflexion points in the scattering curves of the first transitions observed upon heating (T_agg = mid-aggregation temperature obtained from scattering curves). ΔT_agg was calculated in the same way as done for ΔT_m: ΔT_agg = T_agg mutant – T_agg wild-type.

Concentrated (~10 mg ml) GgKDELR2 was diluted to 0.2 mg/ml into buffer consisting of 10 mM citric acid, 20 mM di-sodium phosphate at pH 5.4, 5.9, 6.4, or 7.0 containing 0.01% DDM:CHS (20:1 ratio). To this 0.5 mM KDEL, RDEL, or HDEL peptide (diluted in water) was added, or water as a control. The sample was incubated at room temperature for 15 min. Thermal measurements were carried out in a range from 20°C to 90°C with 1°C/min steps using a Prometheus NT.48. The PR.ThermControl (NanoTemper) software was used to calculate the melting temperature for each condition. The data shown in the manuscript is the calculated melting temperature for each peptide at the given pH with the T_m for the water control at the same pH subtracted.

For Nanoscale Differential Scanning Fluorometry (NanoDSF) measurements, a Prometheus NT.48 (NanoTemper Technologies GmbH, Munich, Germany) was used to analyze protein thermal stability in response to ligand binding. Purified recombinant human NLRP3_ΔPYD (1 µM) was incubated at room temperature with and without 100 µM of MCC950 + 1 mM ATP (Sigma A2383) in 12 µL reaction volume for 30 min in reaction buffer containing 25 mM HEPES pH 7.4, 400 mM NaCl, 0.2% CHAPS, 0.2 mM TCEP. Assays were prepared in Greiner 384 well non-binding black plates (Greiner 784900) and transferred to the instrument using the Prometheus NT.Plex nanoDSF Grade Standard Capillary Chips (PR-AC002). The instrument excites samples at 280 nm and records intrinsic protein fluorescence at 330 and 350 nm, the lambda maxes associated with buried and exposed tryptophan residues, respectively. Measurements were taken over a 20–90 °C thermal gradient with a 1 °C per minute ramp rate. The 350 nm/330 nm fluorescence ratio was plotted versus temperature and the first derivative of this plot was used to determine the melting temperature (T_M) under each condition. Data analysis was done by PR.ThermControl, version 2.1.6 by NanoTemper.