Chirality and biology

Circular dichroism is a consequence of the interaction of polarised light with chiral molecules.

The vast majority of biological molecules are chiral. For instance, 19 of the 20 common amino acids that form proteins are themselves chiral, as are a host of other biologically important molecules, together with the higher structures of proteins, DNA and RNA. The highly chiral chemistry of biological molecules lends itself well to analysis by circular dichroism and the study of biological molecules is the main application of the technique.

A large subset of the use of circular dichroism in biochemistry is in the understanding of the higher order structures of chiral macromolecules such as proteins and DNA. The reason for this is that the CD spectrum of a protein or DNA molecule is not a sum of the CD spectra of the individual residues or bases, but is greatly influenced by the 3-dimension structure of the macromolecule itself. Each structure has a specific circular dichroism signature, and this can be used to identify structural elements and to follow changes in the structure of chiral macromolecules.

The most widely studied circular dichroism signatures are the various secondary structural elements of proteins such as the α-helix and the β sheet. This is understood to the point that CD spectra in the far-UV (below 260nm) can be used to predict the percentages of each secondary structural element in the structure of a protein. Some of the common protein secondary structural elements and the CD spectra associated with them are shown below.

The secondary structure conformation and the CD spectra of protein structural elements. Right is an example of the backbone conformation of a peptide in an α-helix and left is the conformation of a peptide in a β-sheet. In the centre are the associated CD spectra for these different conformations.

 

There are many algorithms designed for fitting the circular dichroism spectra of proteins to provide estimates of secondary structure. The protein secondary structure CD analysis software distributed with the Chirascan is CDNN.

Secondary structure prediction is only part of the power of circular dichroism spectroscopy. Changes in circular dichroism spectra are very good proxies for changes in the structure of a molecule. Couple this with the facts that (i) spectra can be recorded in minutes and (ii) single wavelength kinetics can be recorded from milliseconds onwards, CD is a particularly powerful tool to follow dynamic changes in protein structure. For instance changes induced by changing temperature, pH, ligands, or denaturants are all commonly used.

An application note that describes the use of circular dichroism to follow the kinetics of refolding of the secondary structure of a protein using changes in denaturant concentration can be found here. Another application note that describes the use of circular dichroism to follow the unfolding of proteins by thermal denaturation can be found here.

A powerful application of circular dichroism is to compare two macromolecules, or the same molecule under different conditions, and determine if they have a similar structure. This can be used simply to ascertain if a newly purified protein is correctly folded, determine if a mutant protein has folded correctly in comparison to the wild-type, or for the analysis of biopharmaceutical products to confirm that they are still in a correctly folded active conformation.

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Introduction to Circular Dichroism Understanding circular dichroism: the basics of polarisation Chiral molecules: the origin of optical activity Chirality and biology Principle of operation of a circular dichroism spectrometer Performance of a circular dichroism spectrometer Circular dichroism signatures of secondary structural elements CD units and conversions

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