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 (Tm) 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 Tm.
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 Tm.
Free full text: Click here
