Circular Dichroism

Circular Dichroism Spectroscopy

Circular dichroism spectroscopy is a fast, quantitative and non-destructive photometric technique that can help answer an array of common questions about the stability, structure and interactions of nucleic acids and proteins:

  • has mutation altered the secondary and tertiary structures of a protein?
  • what is the stability of a protein or nucleic acid or their variants
  • does ligand binding cause a change in biomolecule conformation? 

Research questions addressed by CD

  1. Determination of protein secondary structure. 
    Secondary structure can be determined by CD spectroscopy in the "far-UV" spectral region (190-250 nm). At these wavelengths the chromophore is the peptide bond, and the signal arises when it is located in a regular, folded environment. α-helix, β-sheet and random coil structures each give rise to a characteristic CD spectrum (see graph, right). The approximate fraction of each secondary structure type that is present in any protein can thus be determined by analyzing its far-UV CD spectrum as a sum of fractional multiples of such reference spectra for each structural type.  Online tools for protein CD spectra deconvolution such as DichroWeb provide quantitative analysis of secondary structure content or changes in conformation induced by mutation, ligand binding or solution conditions.      
  2. Thermal melts as a measure of stability
    For most proteins secondary structure is lost upon unfolding and the far-UV CD spectra of a folded and unfolded protein are thus distinct.  Measuring the intensity of the CD signal at a fixed wavelength (e.g. 220 nm for an a-helical protein) as a function of temperature gives a measure of thermal stability.  A six-cell changer is available in the facility, which together with an automated temperature ramping method allows the effects of mutation on the stability of a protein and its variants to be assessed rapidly.   
  3. Information about protein tertiary structure
    The CD spectrum of a protein in the "near-UV" spectral region is attributable to phenylalanine (250-270 nm), tyrosine (270-290 nm) and tryptophan residues (280-300 nm).  In a folded protein these aromatic side-chains are static within a chiral environment.  If these residues are in a dynamic region or the protein is unfolded, chirality is lost and no CD signal is observed.  Near-UV CD spectroscopy can thus be sensitive to protein tertiary structure including changes upon ligand binding, protein-protein or protein nucleic acid interactions.
  4. Conformational changes in proteins
    Structural changes in proteins caused by the binding of ligands are an essential part of the mechanism of action and regulation of biological activity. CD provides an experimentally convenient means of detecting such changes which can be examined in different spectral regions. In addition CD can be used to assess the range of ligand concentrations over which structural changes take place (therefore allowing measurement of Kα) and the extent of the changes in the protein of interest and (using time-resolved CD studies) the speed at which such changes occur.
  5. Nucleic acids and other biomolecules
    CD spectroscopy is not limited to proteins.  Any chiral biomolecule or biomolecule held in a chiral environment is amenable to analysis and can yield similar data to those explained above for peptides and proteins.  For example, CD spectroscopy was used to identify the rare Z-form of DNA and to measure the binding of RNA aptamers to their targets.