Circular Dichroism (CD) and Higher Order Structure (HOS) of biomolecules

Most biomolecules are chiral with a higher order structure (HOS) containing chiral chromophores. For example, 19 of the most common amino acids found in proteins are chiral. The combined effect of all chiral elements generates a characteristic CD spectrum, also referred to as a CD signature or fingerprint. This can be used to identify structural elements and to follow changes in the HOS of macromolecules.

The HOS of a protein is particularly sensitive to changes in the environment (such as pH or temperature) and when interacting with other molecules such as ligands, denaturants or nanoparticles. Amino acid composition as well as glycosylation or oxidation states, amongst others, will also affect HOS.

Changes seen in the CD fingerprint of a protein are therefore indicative of changes in secondary and/or tertiary structure. These changes can, in turn, affect characteristics such as protein stability or biological activity or reveal the folded/unfolded state of a protein.

Although suitable for studying chiral molecules of all types and sizes, Chirascan CD spectrometers have been optimized to provide insights into secondary and tertiary structure, as well as kinetic and thermodynamic information, when studying proteins and other chiral biomolecules.

The sensitivity of a Chirascan CD spectrometer reveals similarities and differences when comparing the same molecule under different conditions or comparing different molecules. For example, CD analysis can ascertain if a newly purified protein is correctly folded, determine if a mutant protein has folded correctly in comparison to the wild-type, or enable comparison of biotherapeutic candidates during drug development.

Secondary structure

Measuring CD over the far-UV range (180 nm – 250 nm) generates a CD fingerprint of the secondary structure of biomolecules. These structures typically contain asymmetric motifs such as α-helices and β-sheets. The presence of disulphide bonds may also influence the final CD fingerprint.

Far-UV CD spectra of proteins reveal important characteristics about their secondary structure such as the proportion of α-helix and β-sheet.

Secondary structure, far-UV

Human insulin: signals from peptide backbone dominate in CD spectrum.


Schematic illustrates a hexamer of insulin molecules showing α-helices

Monoclonal antibody (mAb): signals from peptide backbone dominate the CD spectrum. As expected, the secondary structure of this NISTmAb is predominantly β-sheet.

Schematic illustrates a typical antibody showing β-sheet content

Tertiary structure

Measuring CD over the near-UV range (>250 nm) generates a CD fingerprint of the tertiary structure of biomolecules. The fingerprint is influenced by the nature of the surrounding environment of aromatic side chains of the amino-acids tryptophan, phenylalanine and tyrosine. The presence of disulphide bonds may also influence the final fingerprint.

Near-UV CD spectra are not assigned to a specific 3D structure, but do provide structural information.

NIST monoclonal antibody reference material

Differences in near-UV spectra of two mAbs show sensitivity of CD to slight changes in orientation of aromatic side chains

Kinetic and thermodynamic information

The speed of Chirascan CD spectrometers, recording spectra in minutes and single wavelength kinetics from milliseconds onwards, enables HOS changes in secondary and tertiary structure to be followed dynamically, for example, when changing temperature, pH or the concentration of denaturing agents such as guanidinium chloride or urea.

CD is frequently used to determine the effects of mutations or interactions on protein stability by following the unfolding and folding of proteins as a function of temperature. Analysis may also reveal the presence of folding intermediates.

As long as the change in CD as a function of temperature is reversible, the data can be used to determine the van’t Hoff enthalpy and entropy of unfolding, the midpoint of the unfolding transition and the free energy of unfolding. The unfolding curves can be used to indicate the binding constants of protein-protein and protein-ligand interactions.

Secondary structure, far-UV, 25-95°C temperature ramp, Chirascan 6-cell turret, Chirascan

Secondary structure, far-UV, 25-95°C temperature ramp, Chirascan 6-cell turret, Chirascan