Prometheus nt 48

Thermal-shift assays of SARS-CoV-2 Mpro and its H172Y mutant were carried out using the nanoDSF method as implemented in the Prometheus NT.48 (NanoTemper Technologies). The nanoDSF method is based on the autofluorescence of tryptophan (and tyrosine) residues to monitor protein unfolding. As the temperature increases, the protein will unfold and the hydrophobic residues of the protein get exposed, the ratio of autofluorescence at wavelengths 350 nm and 330 nm will change. The first derivative of 350/330 nm can be used to determine the melting temperature (Tm). 30 µM of WT or mutant protein were diluted in a final volume of 15 µL reaction buffer containing 20 mM HEPES, 120 mM NaCl, 0.4 mM EDTA, 4 mM DTT, 20% glycerol, pH 7.0. Then the proteins were loaded onto Prometheus NT.48 nanoDSF Grade Standard Capillaries (PR-C002, NanoTemper Technologies), the fluorescence signal was recorded under a temperature gradient ranging from 25 to 90°C (incremental steps of 0.5°C min⁻¹). The melting curve was drawn using GraphPad Prism 7.0 software; the values of the first derivative of 350/330 nm were displayed on the Y axis. The melting temperature (Tm) was calculated as the mid-point temperature of the melting curve using the PR.ThermControl software (NanoTemper Technologies).

Construction of Thc_cut1 and Thc_cut2 genes, sequencing, protein expression and purification by Ni‐affinity chromatography were performed using methods described previously (Herrero Acero et al., 2013 (link); Ribitsch et al., 2017 (link)). Molar enzyme concentrations were determined by Abs280 and the calculated extinction coefficients (Gasteiger et al., 2005 ). The thermal transition mid‐point (T_m) was determined by differential scanning fluorimetry using a Nanotemper Prometheus Nt.48 (Nanotemper). Enzyme samples (in phosphate buffer) were heated from 20°C to 99°C at 10% laser intensity and a rate of 1.5°C/min. Resulting melt curves are found in Figure S3. Activities at different pH values were determined for both enzymes in the pH range of 6–8.7. For this, volumetric activities (1 µmol of pNP released per minute and ml) at each pH was measured using 3.125 mM pNP‐C8 as substrate and 50 mM sodium phosphate (pH 6–8) and 50 mM Tris‐HCl (pH 8 and 8.7) as buffers. pH profiles are depicted in Figure S4.

The stability of lysozyme was confirmed by nano differential scanning fluorimetry (Nano DSF) (NanoTemper Prometheus NT 4.8, NanoTemper Technologies GmbH, München, Germany), which is based on the intrinsic fluorescence of tryptophan residue at 330 and 350 nm, located in the hydrophobic core of lysozyme. For this purpose, we investigated the stability of lysozyme by a change in melting point toward different concentrations of processing materials, e.g., sonication, acetone, acetate, PBS, PDDMA, and SpAcDEX. All samples were centrifuged for 5 min, at 13,000 rpm at 4 °C and placed in high-sensitivity glass capillaries (Cat#PR-C006, NanoTemper Technologies, GmbH, München, Germany). The measurement temperature was set in the range (0–95 °C), and the heating rate was (2 °C/min). The result is reported as the melting temperature (Tm), the temperature where half of the protein is unfolded. By employing the Therm Control Software V 2.1.5, the fluorescence intensity ratio (F350/F330) was plotted against different temperatures, and the inflection point (IP350/330) of the transition was calculated from the maximum of the first derivative of each data point.

Thermal denaturation was followed using intrinsic protein fluorescence measured with the NanoTemper Prometheus NT48 instrument (Nanotemper Technologies, München, Germany). Samples in HBS-P (Cytiva) supplemented with 5% (v/v) DMSO containing 3 μM full length p110α/p85α and a 1:2 dilution series of 1938 (from 440 μM to 13.8 nM) were loaded into standard capillaries and heated at 2 °C/min from 15 to 95 °C. The first derivative of the fluorescence emission ratio 350/330 nm were analyzed using the PR.ThermControl v2.3.1 (NanoTemper), to define the Tm. Independent experiments using the same protein and compound stocks were performed in triplicate. Data were fitted using Prism 9.4.1 (GraphPad Software Inc). Dissociation constants were calculated using fits to a single-site ligand depletion model: $$T=T_0+\frac{(T_1-T_0)\{([C_T]+[P_T]+[K_D])-\sqrt{{([C_T]+[P_T]+K_D)}^2-4[C_T][P_T]\}}}{2[P_T]}$$ where T₀ and T₁ are the T_m in the absence of titrating compound and at saturation respectively, [P_T] and [C_T] are the total concentrations of protein and compound respectively and K_D is the dissociation constant.

Thermal stabilization assays were performed using the NanoTemper Prometheus NT48 instrument (Nanotemper Technologies, München, Germany). All experiments were performed in 150 mM NaCl, 50 mM Tris pH 8, 1 mM DTT and 1% v/v DMSO unless otherwise noted. Trim7 PRYSPRY was used at 10 μM and peptides at 90 μM. Samples were heated at 2 °C/min from 15 to 95 °C. Data were analyzed using the NanoTemper Prometheus Control Software, and the first derivative of the melt curves used to define the Tm for the apo and complexed samples. Independent experiments used the same peptide and protein stocks.