J 720 spectropolarimeter

Circular dichroism (CD) measurements were performed with a Jasco J720 spectropolarimeter (Jasco, Inc., Easton, USA). Data of absorbed wavelength from 190 to 250 nm were collected within a phosphate buffer (pH 6.0) using the following instrument settings (for an average of 30 scans): response, 1 s; sensitivity, 100 mdeg; speed, 50 nm/min, average of 30 scans. The protein concentration was ~3 μM (~0.1 mg/mL) in 100 mM potassium phosphate buffer (pH 6.0). The CD data was further deconvolved by K2d method (Dichroweb server) to determine the content of secondary structures. The thermal denaturation was carried out by observing the impact of temperature on protein secondary structure. The protein samples were heated from 20 °C to 90 °C and then cooled to 20 °C by a Jasco programmable Peltier element with a heating/cooling rate of 1 °C/min. The wavelength of 209 nm that characterized the α-helix within protein was used to monitor the unfolding rate of the protein structures. The proteins were diluted to 0.1 mg/mL within 5 mM potassium phosphate buffer (pH 6.0/7.0) which had been conducted for a heating study to observe pH shift. The solvent contribution was discounted to correct the obtained spectra. The denaturation temperature (T_m) was calculated according to the maximum slope that signified the swift unfolding of secondary structures.

All solvents and reagents were of analytical grade and were obtained from commercial sources. The UV spectra, optical rotations, and electronic circular dichroism (ECD) spectra were recorded using a Beckman DU 530 Life Science UV/Vis spectrophotometer (Beckman Coulter, Brea, CA, USA), HORIBA SEPA-300 high sensitive polarimeter (HORIBA, Kyoto, Japan), and JASCO J-720 spectropolarimeter (JASCO, Tokyo, Japan), respectively. Infrared spectra were recorded using a HORIBA FT-720 IR spectrometer equipped with a DuraSampl IR II ATR instrument. NMR spectra were recorded using a JEOL ECA-500 FT-NMR spectrometer at 500 MHz for ¹H NMR and at 125 MHz for ¹³C NMR (JEOL, Tokyo, Japan). Chemical shifts were reported in ppm and referenced to the residual chloroform signals (δ_H 7.24 and δ_C 77.23 ppm). High-resolution electrospray ionization time-of-flight mass spectrometry (HRESITOFMS) was performed using a Waters Synapt GII (Waters, Milford, MA, USA). Medium-pressure liquid chromatography was performed using a Teledyne ISCO CombiFlash Companion (Teredyne ISCO, Lincoln, NE, USA). Preparative HPLC was performed using a Waters 600E pump system equipped with a Cosmosil C18 MS-II or AR-II column (Nacalai Tesque, Kyoto, Japan).