Circular dichroism (CD) is defined as the differential absorbance of left circularly polarized light (LCPL) and right circularly polarized light (RCPL): CD = Abs (LCPL) – Abs (RCPL). To be “CD active”, a molecule must be structurally asymmetric and exhibit absorbance. Asymmetry can result from chiral molecules such as the peptide backbone of proteins, a non-chiral molecule covalently attached to a chiral molecule (aromatic amino acid side chains), or a non-chiral molecule in an asymmetric environment (e.g., a chromophore bound to a protein). Increased relative absorption of left polarized light results in a positive CD signal, while a negative signal is the result of right polarized light being more highly absorbed. The most commonly studied molecules are proteins. Proteins are CD active (all amino acids except glycine contain a chiral carbon, thus are asymmetrical), and the resulting CD signals are sensitive to protein secondary and tertiary structure.

Three common secondary structure motifs (alpha-helix, beta sheet, and random coil) exhibit distinctive CD spectra in the far-ultraviolet region (170-260 nm). Using CD spectra, secondary structure of proteins can be estimated using a variety of computer algorithms. The near ultraviolet region (320-260 nm) provides a fingerprint of the tertiary structure of proteins. Asymmetric environments of aromatic amino acids, which are sensitive to protein conformation, provide the basis of the near-UV CD signal. CD is commonly used in denaturation experiments in which the CD signal of a protein is monitored while the protein is perturbed in some fashion (e.g., increasing temperature or chemical denaturant). Changes in CD signal reflect changes in the protein structure. Information about protein stability or folding intermediates can be obtained. In addition to the ultraviolet region, structural information from visible region can be obtained as well in proteins containing chromophores (e.g., hemes) (“A Practical Guide to Using the Olis CD”, Dr. P. Boxrud, Olis Staff Scientist).

CD is reported in units of absorbance or ellipticity. Each of these can be normalized for molar concentration of the sample. The most direct data from the Olis CD instrument is absorbance (Abs[L] – Abs[R]). This value is typically reported in miliabsorbance units (mA), which are a thousandth of an absorbance unit. CD data are also reported as ellipticity (θ), which is related to absorbance by a factor of 32.98 θ = 33.98 ΔAbs). Ellipticity is usually reported in millidegrees (mdeg or m°), which are a thousand of a degree. Molar ellipticity ([θ]) is CD corrected for concentration. The units of molar elliplicity are historical (deg cm²/dmol). Conversion from molar extinction (absorbance corrected for concentration) to molar ellipticity uses a factor 3298 ([θ] = 3298Δε). To calculate molar ellipticity, the sample concentration (g/L), cell pathlength (cm), and the molecular weight (g/mol) must be known. If the sample is a protein, the mean residual weight (average molecular weight of the amino acids it contains) is used in place of the molecular weight, essentially treating the protein as a solution of amino acids.

CD spectra in the far-UV region (170-260 nm) provide important information about protein secondary structure. Common secondary structure motifs exhibit predictable CD spectra. Based on these and the spectra of standard proteins, there are many algorithms currently available for protein secondary structure analysis. The one that is available in Globalworks is based on the algorithm shown in Analytical Biochemistry (Compton, L.A. and Johnson, C.W., Analytical Biochemistry. 155-167, 1986). This algorithm uses a basic set of sixteen model protein structures. The shape of the spectrum is compared to the basis spectra and five structural contributions are extracted (alpha-helix, parallel beta-sheet, antiparallel beta-shit, beta turns, and other). To collect data for this analysis, a rather rigid format must be adhered to. The data must start at 260 nm and span 2 nm per data point, and the data must end at 184, 182 or 180, or 178 (38-41 data points). No information about protein concentration is required. Mention of secondary structure prediction algorithms may be found at the following sites:

http://akilonia.cib.csic.es/~pablo/K2D)

If the data recorded in molar ellipticity, the alpha-helical content can be estimated from the molar ellipticity at 222 nm by equation (Morrow, J.A., Segal, M.L., Lund-Katz, S., Philips, M.C., Knapp, M., Rupp, B. and Weigraber, K.H. Biochemistry. 39, 11657-11666, 2000. see (“A Practical Guide to Using the Olis CD”, Dr. P. Boxrud, Olis Staff Scientist).

One of the most useful applications of CD in the study of proteins is monitoring protein denaturations, which can be initiated either thermally or chemically. In the experiment, CD data are collected as a function of temperature or denaturant concentration. Data can be collected at a single wavelength, resulting in two-dimensional denaturation curves, in which CD signal is recorded versus temperature or denaturant concentration. These curves are fit by known denaturation models to give information about protein structure and stability. Additionally, CD spectra can be collected as a function of temperature or denaturant, resulting in a three-dimensional data set with axes being wavelength, temperature or concentration, and CD intensity. The advantage of this is that data from many wavelengths are included in the subsequent fitting procedures. This 3D data set should be passed though singular value decomposition (SVD) to determine the number of species involved in denaturation and to remove the noise so that the data can be better fitted to unfolding mechanisms to obtain thermodynamic information.

Please contact Krystyna Brzezinska (kbrzez@mrl.ucsb.edu) to schedule training. Before training starts please read  MANUAL