Conformational Analysis of CrataBL Protein

CD spectra were obtained with a J-810 JASCO spectropolarimeter. Measurements were carried out at 25 °C at a CrataBL concentration of 10 μM in a 1 mm pathlength cuvette and were recorded in the 190–250 nm range as an average of eight scans. The results were expressed as the mean residue ellipticity, [θ], defined as [θ] = θ_obs/(10.C.l.n), where θ_obs is the CD in millidegrees, C is the protein concentration (M), l is the pathlength of the cuvette (cm), and n is the number of amino acid residues (165, as described by Ferreira et al. (2013)^{22 (link)}). The CDPro software was used to estimate the fractions of the secondary structure^{28 (link)} and the Cluster program was used to determine the tertiary structure class.^{29 (link)}The effect of pH on the CrataBL conformation was determined by CD. CrataBL, at an initial concentration of 2.0 mg/mL, was diluted with 10 mM acetate/phosphate/borate (PBA buffer), pH 2.0; 4.0; 5.5; 6.0; 7.4; 8.0; 10.0; and 12.0 to a final concentration of 0.2 mg/mL. The protein was incubated for six hours at room temperature.