Melting Temperature

Stability/Folding

Melting Temperature (Tm)

°C (Celsius)

Circular Dichroism (CD)

10 mM sodium phosphate

10 to 90°C

8.0

0.2 mg/mL

222 nm

None

CD measurements were carried out using a Chirascan CD spectrometer (Applied Photophysics model) equipped with a Peltier temperature-controlled cell holder. Experiments were performed at 23°C in a 0.1 cm pathlength cuvette using 0.2 mg/mL protein in 10 mM sodium phosphate buffer (pH 8.0) supplemented with 50 μM ZnSO4. Data were recorded from 260 to 185 nm at 0.5 nm intervals; each data point was averaged for 1 s. Spectra were baseline corrected and smoothed using the Savitzky–Golay filter. Data recorded in the 190–240 nm range were analysed using DichroWeb; (38) the CDSSTR deconvolution method was used to estimate secondary structural content using reference set 4. (39) To minimize the effects of differences in protein concentration, the data were normalized at 207 nm. (40)

Thermal denaturation profiles were monitored by CD at 222 nm, with data recorded every 1°C from 10 to 90°C at a ramp-rate of 1°C/min. Normalized data were fitted to a Boltzmann sigmoidal curve in GraphPad Prism® 5.01 software to determine melting temperature values. Spearman's rank correlation coefficient was used to compare the data from DSF with the temperature-dependent CD results to determine their correlation (Supplementary Data). The correlation analysis was carried out using StatsDirect (http://www.statsdirect.com/).

References:
38 Whitmore L Wallace BA Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases Biopolymers 2008 89 392 400

39 Sreerama N Woody RW Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set Anal Biochem 2000 287 252 60

40 Raussens V Ruysschaert JM Goormaghtigh E Protein concentration is not an absolute prerequisite for the determination of secondary structure from circular dichroism spectra: a new scaling method Anal Biochem 2003 319 114 21