J 810 circular dichroism spectropolarimeter

Circular dichroism (CD) spectra were conducted with a J-810 Circular Dichroism Spectropolarimeter (Jasco, Japan) equipped with a microcomputer, the apparatus was sufficiently purged with 99.9% dry nitrogen gas before starting the instrument and then it was calibrate with d-10-camphorsulfonic acid. All the CD spectra were gathered at 298 K with a PFD-425S Peltier temperature controller attached to a water bath with an accuracy of ±0.1 °C. Each spectrum was recorded with use of a precision quartz cuvette of 10 mm path length and taken at wavelengths between 200 and 260 nm range that provides a signal extremely sensitive to small secondary conformational disturbances. Every determination was the average of five continual scans encoded with 0.1 nm step resolution and got at a speed of 50 nm min⁻¹ and response time of 1.0 s. All observed CD data were baseline subtracted for buffer and the evaluation of the secondary structure components was gained by employing Jasco Spectra Manager II, which calculates the different designations of secondary structures by comparison with CD spectra, determined from diverse proteins for which high-quality X-ray diffraction data are available.^{46 (link)}

Conformational studies of EcCI (0.1 mg mL⁻¹⁾ were carried out in a Jasco J-810 circular dichroism spectropolarimeter at 25 °C in a 1 mm path length cuvette. The CD spectrum was recorded in the 190–250 nm range as an average of eight scans. Data were expressed as the mean residue ellipticity [θ] [29 (link)]. The percentage of secondary structure was estimated by deconvoluting the CD spectrum using the CDPro software package, which contains three CD analysis programs: CONTINLL, SELCON3, and CDSSTR with a protein reference set of 37 proteins [30 (link),31 (link)].
To analyze the pH effect on the secondary structure of EcCI, the protein (0.1 mg mL⁻¹) was incubated in phosphate-borate-acetate buffer (PBA) at 10 mM during 3 h and pH values of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0. CD measurements were recorded as describe above [29 (link)].
The effect of temperature on the secondary structure of EcCI was also analyzed. Protein samples in 10 mM Tris/HCl buffer pH 8.0 (0.1 mg mL⁻¹) were heated at 25, 40, 60, 80, and 100 °C for 2 h. The CD spectrum in the 195–200 nm UV range was recorded as described above.