What is Circular Dichroism?
Circular dichroism is an optical phenomenon that arises from the interaction of chiral molecules with circularly polarized light. It refers to the difference in absorbance of left- and right-circularly polarized light by a sample. Chirality is a property of molecules that are non-superimposable on their mirror image. In the world of biomolecules, many such molecules, including proteins, nucleic acids, and carbohydrates, exhibit CD activity due to their chiral nature.
How Does Circular Dichroism Work?
CD spectroscopy is based on the differential absorption of left- and right-handed circularly polarized light by a sample. Circularly polarized light is a type of light in which the electric field vector rotates in a circular pattern as the light propagates through space. The direction of rotation of the electric field vector can either be clockwise or counterclockwise, and this is referred to as the circular polarization of the light.
When a chiral molecule, such as a protein, interacts with circularly polarized light, it preferentially absorbs one of the two circular polarization components, resulting in a difference in absorption between left- and right-handed light. This difference in absorption can be measured as a function of wavelength to produce a CD spectrum.
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The magnitude and shape of the CD spectrum provide valuable information about the structural characteristics of the molecule being studied. For example, proteins that contain predominantly alpha-helical secondary structure typically exhibit a negative peak in the CD spectrum around 222 nm and a positive peak around 208 nm, whereas proteins that contain predominantly beta-sheet secondary structure typically exhibit a negative peak around 218 nm and a positive peak around 195 nm. Other types of secondary structures, such as random coils and turns, exhibit a low level of ellipticity across the entire spectrum.
The CD spectrum is affected by a variety of factors, including the structure and composition of the molecule being studied, the solvent environment, and the pH and temperature of the sample. Additionally, the CD spectrum can be influenced by the presence of ligands or other small molecules that bind to the protein, as well as by the presence of other proteins or macromolecular assemblies in the sample.
Circular Dichroism Spectrometer and Detector
To perform CD spectroscopy, a specialized spectrometer and detector are required. The CD spectrometer is typically comprised of three main components: a light source, a sample compartment, and a detector. Circularly polarized light is emitted from the light source, and this light passes through the sample compartment containing the sample. The detector then measures the difference in absorbance of left- and right-circularly polarized light by the sample.
CD spectrometers are available in a wide range of configurations, from simple manual instruments to fully automated systems that offer advanced features such as temperature control and multiple sample compartments. The detector used in CD spectroscopy is usually a photomultiplier tube or a diode array detector, both of which are highly sensitive and capable of accurately detecting even subtle differences in the absorbance of left- and right-circularly polarized light.
Circular Dichroism Spectroscopy (Hoffmann et al., 2016)
Circular Dichroism Interpretation
The interpretation of CD spectra requires an understanding of the relationship between the structure of the molecule and the CD signal. For proteins, the CD spectrum is dominated by the contributions from the secondary structure elements, such as alpha-helices and beta-sheets. The CD signal arises from the differential absorption of left- and right-circularly polarized light by chromophores in the molecule.
The alpha-helix structure gives a characteristic negative band at 208 nm and a positive band at 222 nm, while the beta-sheet structure gives a negative band at 216 nm and a positive band at 195 nm. Other structures, such as unordered or disordered regions, have a weak CD signal and give a relatively flat spectrum.
The interpretation of the CD spectra can be aided by using software to fit the experimental spectra to models of protein structure, such as the CONTINLL program.
CD spectra of polypeptides and proteins with representative secondary structures (Greenfield et al., 2006)
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References
- Hoffmann, Søren Vrønning, Mathias Fano, and Marco van de Weert. “Circular dichroism spectroscopy for structural characterization of proteins.” Analytical Techniques in the Pharmaceutical Sciences (2016): 223-251.
- Greenfield, Norma J. “Using circular dichroism spectra to estimate protein secondary structure.” Nature protocols 1.6 (2006): 2876-2890.