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Circular dichroism (CD) spectrometry

PB Paul J. Bujalowski
WB Wlodzimierz Bujalowski
KC Kyung H. Choi
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The far-UV circular-dichroism measurements for wild-type and mutant proteins were performed on a model No. J-815 spectrometer (JASCO, Oklahoma City, OK). The protein concentration was ~ 0.1 mg/ml in 50 mM Tris buffer (pH 7.4) containing 150 mM NaCl, 10% glycerol and 1 mM MgCl2. CD spectra were collected at 20 °C in a 0.2-cm path-length cuvette from 195 to 260 nm. Three scans were averaged in the range of 200–250 nm. Data were converted from millidegrees to mean residue ellipticity (MRE) using the formula:

where [θ] is the molar residue ellipticity, θ is the CD signal in millidegrees, c is the protein concentration in µM, l is the path length in mm, and n is the number of amino acid residues. The CD data analysis program BeStSel was used to analyze the data and estimate the secondary structure41.

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