Figure 5
Secondary structure analysis by CD-spectroscopy.
Overlay of far-UV CD spectra of un-autoclaved and autoclaved enzymes (a). CD spectra were determined for each protein at a concentration of 0.2 mg/ml in 50 mM potassium phosphate buffer (pH 7.8) using a Jasco-815 spectropolarimeter (JASCO, Japan) maintained at 4°C. Far-UV (190–260 nm) CD spectra of protein was recorded in a quartz cuvette of 0.1 cm path length at a scan speed of 50 nm/min, with a 1 nm bandwidth, 1 nm data pitch and a 1s response time. Solid (blue) and dashed (pink) lines represent un-autoclaved and autoclaved enzymes, respectively. Far-UV CD spectra of WT and mutant enzymes as a function of temperature is shown in panel “b”. CD spectra (190–260 nm) were recorded over a temperature range of 0–100°C in 50 mM potassium phosphate buffer (pH 7.8 ) in a quartz cuvette of 0.1 cm path length at a scan speed of 50 nm/min, with a 1 nm bandwidth, 1 nm data pitch and a 1s response time. Spectra were analyzed every 10°C by incubating the protein (0.2 mg/ml) at defined temperature for 5 min for a temperature range of 0–100°C. WT, wild type; C56A, cysteine at position 56 substituted with alanine; C95A, cysteine at position 95 substituted with alanine; C145A, cysteine at position 145 substituted with alanine.
