Microcal vp dsc calorimeter

Seeds were obtained from the preformed fibrils of heat-induced spontaneous amyloid formation under stirring and were moderately sonicated before seeding experiments. In the case of ThT assays, the conditions for sample preparation and measurements were the same as for standard experiments, except for the addition of 5% (v/v) seeds.
Calorimetric measurements were carried out using a MicroCal VP-DSC calorimeter (Malvern Panalytical, Ltd, Worcestershire, UK)^{63 (link)}. The sample solution contained 0.5 mg/ml of each protein and the solution conditions were the same as those for the individual ThT assays with or without seeds. Sample and buffer solutions were carefully loaded into the DSC sample and reference cells, respectively, after being properly degassed in an evacuated chamber for 3 min at 25 °C. After the buffer–buffer baseline was subtracted from the sample data, apparent heat capacity (Cp) corresponding to the whole sample solution was evaluated using ORIGIN software (Microcal Inc.).

For RibR and Rib2R, protein samples (1 mg/mL in 20 mM NaH₂PO₄, pH 7.4) and well-matched buffer were degassed for 10 min at 10 °C using a Thermovac degasser (MicroCal). Differential scanning calorimetry (DSC) scans were acquired on a MicroCal VP-DSC calorimeter (Malvern Panalytical) with a scan rate of 90 °C/h. The reversibility of thermal unfolding was verified by repetitive scans on the same sample. A buffer–buffer scan was subtracted from each protein scan, followed by normalization for protein concentration and subtraction of a progress baseline for sample baseline correction. Melting temperatures (T_ms) were derived by fitting the unfolding endotherm using a 2-state unfolding model for data above 20 °C. Data analysis was performed using MicroCal Origin 5.0.
The T_m values for RibL and RibS were determined using a Prometheus (differential scanning fluorimetry [DSF]) instrument (NanoTemper) at a protein concentration of 1 mg/mL in 20 mM Tris, 150 mM NaCl, pH 7.5, using a sample volume of 10 µL. Samples were heated to 95 °C at a rate of 1 °C/min to generate an unfolding profile.

DSC measurements were performed on a MicroCal VP-DSC calorimeter (Malvern Panalytical). Protein samples were prepared in 20 mM potassium phosphate or HEPES buffer, pH 6.0, 0.2 M NaCl, 10 mM 2-mercaptoethanol, 10% glycerol and 0.02% DDM. A scan rate of 1 °C/min and a protein concentration of 0.5 mg/ml were fixed for all DSC experiments. All thermodynamic data are given per mole of protein. The excess molar heat capacity function was obtained after a baseline subtraction, assuming that the baseline is given by the linear temperature dependence of the native-state heat capacity³⁷ . Buffer–buffer scans were recorded under the same conditions and subtracted from sample endotherms. The denaturation temperature, Tm, corresponds to the maximum of the DSC peak,the total denaturation enthalpy, ΔH, is the integrated area under the DSC peak. The uncertainty was 0.2 °C on the temperature and within 10% on the enthalpy.