Microcal vp dsc instrument

Differential scanning calorimetry (DSC) was carried out on a Microcal VP-DSC instrument from Malvern Panalytical Ltd (Malvern, United Kingdom) with the following set-up: 25–90 °C temperature gradient, 90 °C/h scan rate, 10 min pre-scan equilibration [67 (link),68 (link)]. A solution of 0.6 mg/mL of enzyme was incubated in suitable buffer: 50 mM KPi, 200 mM KCl, 10% glycerol pH 7.0 for ARO constructs; 50 mM KPi, 200 mM KCl, 10% glycerol pH 8.0 for 3A4 constructs. All experiments were conducted with a dilution factor of the storage buffer of 10⁶ of the storage buffer. The denaturation temperature Tm and the ΔH was determined by fitting the data with the ORIGIN embedded program suite. In order to perform the analysis at different scan rates, the cells were pre-equilibrated with at least 10 cycles of buffer in the same setup of the experiment. Data analysis was performed using Microcal Origin software (Malvern, United Kingdom). Cycles of cooling and reheating of the samples were performed to obtain the background for buffer subtraction or test hypothetical refolding. Replicate runs did not vary more than 0.25 °C. None of the constructs exhibited even partial re-folding upon cooling and re-heating the sample indicating full irreversibility of the unfolding processes.

Samples of approximately 5 µM NDs of different types (and if stated 10 µM αS) in running buffer (20 mM sodium phosphate pH 7.4, 50 mM NaCl) were degassed for at least 20 min at 30 °C and measured in a Microcal VP-DSC instrument (Malvern Instruments). After equilibration and a pre-scan delay of 20 min, the thermograms were acquired up-scan from 5 °C to 45 °C at a scanning rate of 0.5 °C min^–1 and corrected by subtracting the thermogram of buffer and adjusting the baseline to zero at 45 °C (y-axis molarity refers to estimated amounts of lipids).