CD spectroscopy to measure protein secondary structure

Far-ultraviolet (UV) circular dichroism (CD) spectra were collected to monitor protein secondary structure. Samples were placed in a 0.5 mm path-length quartz cuvette and spectra were recorded from 190 nm to 260 nm for solutions containing 2 mM and 10 mM KCl (pH 6.0). Samples with 90 mM KCl were monitored from 195 nm to 260 nm to keep the high-tension voltage below 600 V (74). Triplicate samples were analyzed, and 10 scans per sample were measured using a Chirascan-plus spectrometer (Applied Photophysics, Leatherhead, United Kingdom). After subtraction of a blank solution spectrum from the raw average signal, the spectra were normalized by calculating the mean residue ellipticity:

where [θ]_obs is the observed ellipticity, residues is the number of amino acid residues per protein molecule, and L is the cell path length.