Vp capillary dsc instrument

The thermal transition points (T_m) of the NFL trimeric proteins were determined by differential scanning calorimetry (DSC) using a MicroCal VP-Capillary DSC instrument (Malvern). Prior to the DSC melting scan, the protein samples were extensively dialyzed in PBS, pH 7.4, and the concentrations were adjusted to 0.25 mg/ml. The dialysis buffer was used as the reference solution. The DSC experiments were done at a scanning rate of 1 K/min under 3.0 atmospheres of pressure. DSC data were analyzed after buffer correction, normalization, and baseline subtraction using MicroCal VP-Capillary DSC analysis software provided by the manufacturer.

Thermal shift assays for metal binding were carried out in 96‐well format in a Stratagene MX3005P real‐rime PCR machine. 25 μl reactions contained 12 μM ComEC CTD, 1 mM of the metal salt under investigation, 1:5,000 diluted SYPRO Orange dye (Invitrogen), 18 mM Tris‐HCl pH 8.0, and 135 mM NaCl. Samples were heated from 25°C to 94°C in 1°C increments, allowing 30 s for equilibration at each set temperature before fluorescence readings were taken. The melting temperature of the protein (T_m) was extracted by using TSA‐CRAFT software (Lee et al, 2019 (link)) to fit the data to the Boltzmann equation.
Differential scanning calorimetry measurements were performed on a Malvern VP Capillary DSC instrument. The sample cell contained 18.6 μM ComEC CTD protein in 20 mM Tris‐HCl, 150 mM NaCl and, where appropriate, 1 mM MnCl₂. The temperature was raised from 10°C to 110°C at a rate of 200°C/h. Runs without protein were used for baseline subtraction. Data were analyzed using the instrument software. The baseline was subtracted from the raw data, and the data were normalized to protein concentration. The data were fitted to a two‐state unfolding model to extract the T_m.

Samples were dialysed into NMR buffer [18 mM Na₂HPO₄, 7 mM NaH₂PO₄, 100 mM NaCl (pH = 7.2) using 3.5 kDa MW cut‐off dialysis cassettes (Thermo Scientific)] and subsequently diluted to 50 μM. DSC was performed using a VP‐capillary DSC instrument (Malvern Instruments). Samples were scanned at a heating rate of 90°C/h in mid‐feedback mode. Data were corrected for instrumental baseline using average buffer scans recorded immediately before and after Ub runs and plotted. After concentration normalisation, the intrinsic protein baseline between pre‐ and post‐transitional levels was corrected using the progress function in the Origin software supplied with the instrument. Corrected endotherms were fitted to a non‐two‐state model allowing T_m, ∆H calorimetric and ∆H van't Hoff to vary independently.