Circular dichroism (CD) spectroscopy is a technique that measures the difference in absorption of left-handed and right-handed circularly polarized light by optically active molecules. It is standard for rapid determination of protein secondary structure, monitoring conformational changes, and characterizing ligand binding.
The CD signal arises from the interaction of circularly polarized light with asymmetric chromophores. In proteins, the peptide bond absorbs in the far-UV region (180–250 nm), where the CD spectrum reflects the backbone conformation. α-Helices produce a characteristic spectrum with a positive band at 190 nm and negative bands at 208 and 222 nm. β-Sheets show a positive band at 195 nm and a negative band at 218 nm. Random coil structures have a negative band near 200 nm and a weak positive band at longer wavelengths.
Aromatic side chains contribute CD signals in the near-UV region (250–350 nm), which report on the tertiary structure environment of tryptophan, tyrosine, and phenylalanine residues. Disulfide bonds also contribute in this region. Changes in near-UV CD indicate alterations in the packing of the protein core.
Quantitative analysis deconvolves the CD spectrum into fractional secondary structure content using reference databases and algorithms such as CONTINLL, CDSSTR, and SELCON3. The fitting quality depends on the wavelength range and the reference set used. Results are most reliable when spectra are collected down to at least 190 nm.
CD is also used to monitor thermal and chemical denaturation by following the CD signal at a specific wavelength as a function of temperature or denaturant concentration. The melting temperature is determined from the midpoint of the unfolding transition.
Practical considerations include accurate baseline subtraction, appropriate buffer selection (avoiding high absorbance components like imidazole and DTT), and sample concentration optimization. Typical protein concentrations for far-UV CD are 0.1–1 mg/mL in a 0.1 mm pathlength cell.
In addition to proteins, CD characterizes nucleic acid conformation (A-form, B-form, Z-form), polysaccharide structure, and the absolute configuration of chiral small molecules. Synchrotron radiation CD extends the wavelength range into the vacuum UV for improved structural resolution.
