Peltier temperature controller

Far-UV Circular Dichroism (CD) spectra and thermal stability profiles were obtained for IpaC in the tested purification conditions using a JASCO model J-1500 spectropolarimeter equipped with a 6 sample turret and a Peltier temperature controller (Jasco, Easton, MD). Spectral scans were obtained from 200 nm to 260 nm at 10°C, 0.5 nm spectral resolution, 50 nm/min scan rate, and a 1 sec data integration time in quartz cuvettes with 0.1 cm path length. Thermal stability profiles were generated by monitoring CD signal at 208 nm while the temperature was increased from 10°C to 90°C at 0.3°C/min. All measurements were at an IpaC concentration of 0.5 mg/mL with appropriate detergent concentrations as outlined for each experiment. To mimic experimental conditions, CD data for IpaC in urea was obtained by rapidly diluting concentrated IpaC in 2 M urea to 0.5 mg/mL IpaC and 200 mM urea. Lower urea concentrations were not possible for these measurements due to limitations of IpaC concentration that can be achieved in 2 M urea. All CD signals were converted and reported as mean residue molar ellipticity and the Dichroweb software package K2D was used to analyze secondary structure content.^{31 (link),32 (link)} Thermal transition values were determined by plotting the derivative of each thermal unfolding curve and identifying the derivative maxima corresponding to the transition inflection.

Far-UV CD spectra and thermal stability profiles were obtained for purified monomeric and trimeric Spa47, Spa47^Δ1-6, Spa47^Δ1-79, and MixN. Measurements were collected using a JASCO model J-1500 spectropolarimeter equipped with a six-position sample holder and a Peltier temperature controller (Jasco, Easton, MD). Spectra were collected from 190 to 260 nm at 10 °C using 0.1 cm quartz cuvettes, 0.1 nm data sampling, a 50 nm/min scan rate, and a 2 second data integration time. Secondary structure thermal stability profiles were collected in the same 0.1 cm quartz cuvettes by monitoring the CD signal at 222 nm while the solution temperature was increased from 10 °C to 90 °C at a rate of 0.3 °C/min. CD analysis was performed on 0.5 mg/mL protein for MxiN, isolated monomeric Spa47, Spa47^Δ1-6, and Spa47^Δ1-79, and 0.3 mg/ml for isolated trimeric Spa47. MxiN was evaluated in 20 mM Tris, 100 mM NaCl, 5% (v/v) glycerol, 5 mM DTT, pH 7.9 and all Spa47 samples were in 20 mM Tris, 100 mM NaCl, and 5 mM DTT, pH 7.9. CD signals were converted to mean residue molar ellipticity and secondary structure content analysis was performed using the Dichroweb^{44 (link)} software package K2D.^{45 (link)} Thermal unfolding transition temperatures (Tm) were determined by plotting the derivative of each thermal unfolding curve and identifying the corresponding maxima.