Using circular dichroism spectra to estimate protein secondary structure

Abstract

Circular dichroism (CD) is an excellent tool for rapid determination of the secondary structure and folding properties of proteins that have been obtained using recombinant techniques or purified from tissues. The most widely used applications of protein CD are to determine whether an expressed, purified protein is folded, or if a mutation affects its conformation or stability. In addition, it can be used to study protein interactions. This protocol details the basic steps of obtaining and interpreting CD data, and methods for analyzing spectra to estimate the secondary structural composition of proteins. CD has the advantage that measurements may be made on multiple samples containing < or =20 microg of proteins in physiological buffers in a few hours. However, it does not give the residue-specific information that can be obtained by x-ray crystallography or NMR.

Figure 1

Circular dichroism (CD) spectra of polypeptides and proteins with representative secondary structures. a, CD spectra of poly-L-lysine at pH 11.1 in the 1 (black) α-helical and 2 (red) antiparallel β-sheet conformations and at pH 5.7 in the 3 (green) extended conformations and placental collagen in its 4 (blue) native triple-helical and 5 (cyan) denatured forms. b, CD spectra of representative proteins with varying conformations: 1 (black) sperm whale myoglobin; 2 (green) chicken heart lactate dehydrogenase; 3 (red) bovine α-chymotrypsin and 4 (cyan) human Bence Jones protein REI light chain, which is a human immunoglobulin light chain of κ type. Spectra are from data sets supplied by Dr. W.C. Johnson.

Figure 2. Circular dichroism spectra of lysozyme in 10 mM sodium phosphate pH 7.0

a, The spectra of (black) air; (red) water; (green) buffer and (orange, blue and magenta) three replicate spectra of lysozyme, 0.085 mg/ml in a 0.1 cm cell. b, The spectra of (black, red, green) buffer; and three replicate spectra each of lysozyme at (cyan, purple, brown) 0.41 mg/ml and (blue, magenta, orange) 0.83 mg/ml concentration in a 0.01 cm cell. d, The change in the photomultiplier tube dynode voltage as a function of wavelength for the conditions illustrated in 1a and 1b and for 0.41 mg/ml lysozyme in a 0.1 cm cell. d, The mean residue ellipticity of lysozyme in a 0.1 cm cell (black) 0.41 mg/ml (raw data not shown); (cyan) 0.085 mg/ml; and in a 0.01 cm cell (green) 0.41 mg/ml; (red) 0.83 mg/ml. e, The mean residue ellipticity of lysozyme (black circles) fit using the method of least squares: (blue) using a peptide data base; (black) four basis spectra extracted from 17 proteins1; (orange) five basis spectra extracted from 33 proteins and (magenta ) CONTIN; (cyan) K2D and (orange) SELCON2. F, The mean residue ellipticity of lysozyme (black circles) fit using the CDPro Package: (magenta) SELCON3; (cyan) CDSSTR and (black) CONTIN. Note. Lyoszyme was obtained from Sigma (L6876) and dissolved in sodium phosphate, 10 mM. The protein concentration was determined using the published extinction coefficient of ε_1% of 26.5 (ε_M =38.2 × I0³) for comparison with previous data. Data were obtained on an Aviv Model 215 spectrometer (Aviv Biomedical, Lakewood, NJ).