CD Analysis of WT and Mutant rLifA Proteins

The far ultraviolet (UV) CD spectra of 0.11 µM WT rLifA and rLifA^C1480A were recorded at 10 nm/min; data pitch 0.1 nm; response time 2 s between 185 and 285 nm in a 0.1 cm path length quartz cuvette at 25 °C (Jasco-810 spectropolarimeter). The proteins were exchanged into 10 mM NaH₂PO₄, pH 7.6, 100 mM NaF prior to analysis using a HiTrap desalt column (GE Healthcare) at a flow rate of 4 mL/min. Spectra were corrected by subtracting a buffer baseline, each an average of five spectra, acquired under the same conditions. Secondary structure was estimated using the DichroWeb CD secondary structure analysis server⁵² (link) including the methods CONTIN, SELCON3 and CDSSTR53 (link), 54 (link), 55 (link), 56 (link) and reference data sets 3, 4, 6, 7, SP175 and SMP180.