Paul's* Practical Guide
A Practical Guide to Using the Olis CD
Basics
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)).1 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).
 Fig. 1
(click to open larger image)
Increased relative absorption of left polarized light results in a positive CD signal, while a negative CD 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; Fig 1)2. 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 the visible region can be obtained as well in proteins containing chromophores (e.g., hemes).
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Footnotes
*) Dr. Paul Boxrud, Olis Staff Scientist
1) For an excellent discussion of the properties of circularly polarized light, see: Klinger, D.S., Lewis, J.W., and Randal, C.E. (1990) in Polarized Light in Optics and Spectroscopy pp. 13, Academic Press, San Diego, CA.
2) Copeland, R.A., Methods for Protein Analysis: A Practical Guide, 1994.
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