J 815 cd polarimeter

The secondary structure of AAR was determined by recording far-UV CD spectra on JASCO J-815 CD polarimeter at wavelength 200–250 nm in a 1 mm quartz cuvette. The protein concentration was 3.5 μM in buffer containing 50 mM Tris, 100 mM NaCl, 1 mM DTT at pH 7.4. AAR contains six tyrosine and six tryptophan residues, which help in exploiting the intrinsic fluorescence property of the protein to determine the tertiary structure of the protein via Cary Eclipse fluorimeter. The protein was excited at excitation wavelength of 280 nm and 295 nm with slid width of 10 nm and fluorescence emission spectra were recorded from 300 to 400 nm in 1 cm quartz cuvette. The protein concentration used was 3.5 μM in buffer containing 50 mM Tris, 100 mM NaCl, 1 mM DTT at pH 7.4.

A JASCO J-815 CD polarimeter was used to measure circular dichroism spectra. Samples were typically loaded at concentrations of 0.015–0.15 mg/ml (1–10 μM) in 100 mM KCl, 20 mM CHES, pH 8.58 into a 1 mm pathlength quartz cell. Far-UV CD spectra were recorded at 100 nm/min with a sensitivity of 50 mdeg. To assess the thermal stability of the proteins, a ramp rate of 40 °C/h with 1 °C intervals was used and the ellipticity at 222 nm was measured throughout. The raw data were converted to mean residue ellipticity using the protein concentration and cell pathlength. The thermal denaturation midpoints (T_ms) were assessed by plotting the second derivatives of the denaturation traces, the x-axis intercept corresponding to the T_m.