Circular dichroism analysis

Secondary structure of Est WT and 1-D5 was predicted by using the program PSIPRED [40]. Circular dichroism (CD) spectra of Est WT and 1-D5 were recorded with a Jasco-810 CD spectrometer (Jasco Corp., Japan). The data were collected at room temperature from 195 to 250 nm using 2 mm quartz cuvette (600 μl). The conversion to the Mol CD (∆ε) in each spectrum was performed with the Jasco Standard Analysis software. Estimation of the secondary structure content from far-UV circular dichroism (CD) spectra was performed by using the CDPro software package (available at http://lamar.colostate.edu/~sreeram/CDPro/main.html), including three executable programs (SELCON3, CDSSTR, and CONTIN/LL) [41]. In this study, the percentages of α-helix and β-sheet for each protein sample were averaged by the calculations of results from the CDPro software package. The circular dichroism data were expressed in terms of the mean residue ellipticity (θ_mrw),which calculated with using the equation [42]:

where θ_obs is the observed ellipticity in degrees, Mw is the protein molecular weight of WT and 1-D5, and N is the number of residues, d is the path length of quartz cuvette (0.2 cm), c is the protein concentration (mg/ml), and the constant number 100 stems from the conversion of the molecular weight to mg/dmol.