The British Biophysical Society

Circular Dichroism Spectroscopy

Bonnie Ann Wallace

 

Circular dichroism (CD) spectroscopy is a type of absorption spectroscopy that can provide information on the structures of many types of biological macromolecules. Circular dichroism is the difference between the absorption of left and right handed circularly-polarised light and is measured as a function of wavelength:

CD is measured as a quantity called mean residue ellipticity, whose units are degrees-cm²/dmol. Chiral or asymmetric molecules produce a CD spectrum because they absorb left and right handed polarised light to different extents and thus are considered to be "optically active". Biological macromolecules such as proteins and DNA are composed of optically active elements and because they can adopt different types of three-dimensional structures, each type of molecule produces a distinct CD spectra.

The wavelengths of light that are most useful for examining the structures of proteins and DNA are in the ultraviolet (UV) or vacuum ultraviolet (VUV) ranges (from 160 to 300 nm) because these are the regions of the electronic transitions of the peptide backbone and side chains in proteins and the purine and pyrimidine bases in DNA. Typically the range of data collected on a commercially-available CD instrument will be between 190 and 300 nm.

Different types of protein secondary structures (helices, sheets, turns and coils) give rise to different CD spectra. Because the spectrum of a protein is directly related to its secondary structure content, the spectrum will be a linear combination of each of these "reference" spectra, weighted by the fraction of the type of secondary structure. Therefore, it is possible to mathematically extract the secondary structure information for an unknown protein from its CD spectrum.

CD spectra of proteins with different types of secondarys structures (red=alpha-helical, blue=beta-sheet, yellow=polyprotein-helical).  The area shaded green to the left of the vertical black bar is only accessible by SRCD spectroscopy (see below). (Adapted from Miles & Wallace, 2006)

Some of the advantages of CD spectroscopy are that it requires only very small amounts of material (100 micrograms or less) and measurements can be done very quickly (in 30 minutes or less). CD spectroscopy has been used to monitor: 1) secondary structure, 2) conformational changes, 3) environmental effects, 4) protein folding and denaturation, and 5) dynamics.

The information produced by CD spectroscopy is complementary (See index) to the sorts of structural information obtained by other biophysical methods such as X-ray crystallography and NMR spectroscopy, especially as it can be done under conditions which mimic those found in vivo.

The Future

Synchrotron Radiation sources produce much brighter light than can be obtained from Xenon arc lamps that are used as the light sources in commercial CD instruments. As a result, new developments which have produced a Synchrotron Radiation Circular Dichroism (SRCD) instrument will enable more accurate spectra to be measured to lower wavelengths. One of only two places in the world where such an instrument now exists is at the Centre for Protein and Membrane Structure and Dynamics located at the Daresbury Lab in Cheshire. As a result, it is expected that in the future CD may play an important role in the new area of structural genomics studies. (See Miles and Wallace, 2006.)

This is the spectrum of myoglobin, a mostly helical protein. The blue line is the spectrum obtained with a commercial lab-based CD instrument and the red line is the spectrum obtained with the synchrotron radiation CD instrument at the Daresbury Laboratory, Cheshire (Wallace, 2000).

Reading List

Miles, A.J. and Wallace, B.A. (2006) Synchrotron Radiation Circular Dichroism Spectroscopy of Proteins and Applications in Structural and Functional Genomics. Chem. Soc. Reviews 35:39-51.

Wallace, B.A. (2000) Conformational Changes by Synchrotron Radiation Circular Dichroism Spectroscopy. Nature Structural Biology 7:708-709.

For more information on the physics of CD Spectroscopy, see:

dichroweb.cryst.bbk.ac.uk

www2.umdnj.edu/cdrwjweb/

srs.dl.ac.uk/vuv/home-page/hot-topics/cd.html

Bonnie Ann Wallace is Professor of Molecular Biophysics at Birkbeck College, London. She is also the director of the BBSRC Centre for Protein and Membrane Structure and Dynamics at CLRC Daresbury.