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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