Circularly Polarized Light

Circular Dichroism Units

Chromophores

Applications of CD Spectroscopy

Circularly Polarized Light...

Circularly polarised light can be described in terms of electric (e) and magnetic (m) wave motions. Each wave has magnitude and oscillation properties. The  e wave magnitude is constant and the direction oscillates causing the electric vector to trace a circle.

As the light beam is passed through an optically active sample, the magnitude of e waves will be altered as a result of changes in the molar extinction coefficients of the left and right handed polarized light. The electric vector now traces an ellipse instead of a circle.

Circular Dichroism units...

There are several different units of measurement for circular dichroism. Molar ellipticity, mean residue ellipticity and delta epsilons are all mentioned in the literature. Ellipticity is defined as the tangent of the ratio of the minor to major elliptical axes. More modern CD instruments measure the difference in absorption of right and left circularly polarized light as a function of wavelength. In accordance with the Beer –Lambert law, wavelength is equal to the difference in molar extinction coefficients divided by the product of path length and concentration. Mean residue ellipticity is the most common unit (degree cm2 dmol –1) and delta epsilons are the new machine unit, often referred to as molar circular dichroism (liter mol-1 cm-1), not to be confused with molar ellipticity (degrees decilitres mol-1 decimeter-1).

Chromophores...

Chromophores are optically active groups within the protein. Prime candidates are backbone amide bonds, and disulphide bonds, and aromatic side chains such as Phe, Trp, and Tyr  to a lesser extent. In secondary structure conformations, the backbone and hence the amide bond chromophores are arranged in regular orgranized patterns. CD spectroscopy is extremely sensitive to these patterns and each conformation gives rise to characteristic spectral features.

Absorption bands of protein chromophores:
 

The direct relationship between protein secondary structure and circular dichroism spectra means that circular dichroism spectra can be exploited for prediction of protein conformation.

Sample CD curves:

Prediction of secondary structure via CD spectroscopy gives accuracies of up to 95% for alpha helical proteins. Similar measures for beta sheet or mixed proteins are poor with accuracies of around 50% and 75% respectively. Inaccuracies arise due to the complex multicomponent nature of spectroscopic analysis, considering that spectral effects are governed by all factors influencing protein conformation. It is often impossible to account for all of these factors in statistical anaysis leading to lost information. Other problems stem from ambiguity in  secondary conformational class definition.

The strengths of CD spectroscopy lie in its extreme sensitivity to conformational changes in the protein resulting from changes in the protein solution composition. Particularly the near UV CD region is sensitive to changes in tertiary structure due to protein-protein interactions and changes in solvent conditions. This region picks up noise from disulphide bond chromophores and aromatic amino acids, and this data is useful in thermostability and denaturation studies.

Other applications of CD are in the study of protein folding, and in comparing structures obtained from different species or expression systems. CD analysis is also used in structural prediction of membrane proteins in cases where proteins cannot be crystallised.