Nano dsc instrument

The thermodynamic stability of 3CLpro was measured using a Nano DSC instrument (TA Instruments) calibrated using chicken egg white lysozyme, a known external Nano DSC standard that is part of the TA Instruments test kit (602198.901). The thermogram was acquired at a 3CLpro concentration of 30 μM in 20 mM Hepes (pH 7.0), 150 mM NaCl, and 20% (v/v) DMSO. The protein samples were heated from 15 to 75 °C at a scan rate of 1 °C/min and 3 atm pressure. The background scans were obtained by loading degassed buffer in both the reference and sample cells and heating at the same rate. The DSC thermograms were corrected by subtracting the corresponding buffer baseline and converted to plots of excess heat capacity (C_p) as a function of temperature. The T_m was determined at the maximum temperature of the thermal transition, and the ΔH_cal of the transition was estimated from the area under the thermal transition using Nano Analyzer software (TA Instruments).

Thermal denaturation curves were acquired on a Nano DSC instrument (TA Instruments Inc.). The scan rate was 1 K min⁻¹, and the protein concentration was 1 mg ml⁻¹. The buffer was PBS (137 mM NaCl, 8.1 mM Na₂HPO₄, 2.68 mM KCl and 1.47 mM KH₂PO_4, pH 7.4) containing 0.1 mM TCEP. Reheating experiments were performed in the same manner after heating the protein to the target temperature, followed by gradual cooling to room temperature. Analysis was performed by using CpCalc (TA Instruments Inc.) and data were reported as heat capacity (kJ K⁻¹ mol⁻¹). The transition temperature was defined as the temperature corresponding to the transition peak maximum.

DSC experiments were performed using a Nano DSC instrument from TA Instruments, USA. Prior to DSC scans, solutions of wild type and mutant bRs were prepared by exhaustive dialysis against water or 1 M KCl, pH 7.0. Before the measurements, the sample and reference solutions were properly degassed and carefully loaded into the cell to avoid bubble formation. Subsequently, the samples were introduced into DSC cell holder with a final concentration in the range 1.3–2 mg/ml of bR (the protein concentrations were determined spectrophotometrically). The reference cell was loaded with the aqueous medium obtained from the dialysis of the purple membrane suspension. Data were collected in the temperature range from 298.15 to 383.15 K at a heating rate of 1K/min. After the first heating scan was completed, the samples were cooled to 25°C, and reheated to check transitions reversibility. For each sample, consecutive thermograms were recorded. The DSC data were processed and analyzed using NanoAnalyse software, following the Takahashi and Sturtevant methodology, as previously explained [24 (link)].

Samples were dialyzed in Slide‐A‐Lyzer cassettes (Thermo Fisher Scientific, Waltham, USA) with a molecular weight cut‐off (MWCO) of 10.000 Da to a 20 mM sodium phosphate buffer, pH 6.9 and then diluted to a concentration of 3 mg/ml. The solution was loaded into the sample cell of a TA‐Instruments (New Castle, DE) Nano DSC instrument (model: 602000). The reference cell was filled with a 20 mM sodium phosphate buffer pH 6.9 and a thermoscan from 20 to 100°C with a scan rate of 1°C/min was performed. In between sample runs, the instrument was cleaned by flushing the cells with water and buffer. At the end of each set of experiments, the instrument was cleaned with a solution containing 0.5 M NaCl, 0.1 M acetic acid, and 1 mg/mL pepsin which was incubated for 3 hr at 37°C and then flushed with 2 L of water. The obtained thermogram data were analyzed using the TA Instruments NanoAnalyse software. Buffer blanks were subtracted from all samples.

Differential Scanning Calorimetry (DSC) experiments were carried out using a NanoDSC instrument (TA Instruments). Samples were analyzed at total strand concentrations of  0.18 mg/mL,  0.24 mg/mL and  0.40 mg/mL (i.e. 25 µM, 35 µM and 55 µM, respectively) in a 10 mM PBS buffer pH 6.8. Each sample was heated from 5 °C to 90 °C under an extra nitrogen pressure of 3 atm at a heating rate of 1 °C/min. Samples were heated twice in order to determine the reversibility of the process. Raw DSC curves were corrected for the instrumental buffer-buffer baseline and normalized by strand concentration to obtain molar heat capacity curves Cp(T). Excess molar heat capacities curves (Cp_exc) were obtained from Cp(T), by subtracting a baseline obtained by a fourth-order polynomial fit of the pre- and post-transition Cp trends as described elsewhere^{43 (link),44 (link)}. The number of DSC components to be adopted in the peak deconvolution procedure was selected in order to minimize fitting errors. Cp_exc curves were deconvoluted by the NanoAnalyze software using the Gaussians model. The temperatures (T_m) and enthalpy (ΔH) of strand melting are defined as the temperature at which the Cp_exc curve reaches its maximum value and the area under the Cp_exc(T) peak, respectively.