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CIRCULAR DICHROISM Introduction Circular dichroism (CD) spectroscopy is a type of absorption spectroscopy that can provide information on the structures of many types of biological macromolecules. Biological macromolecules such as proteins and DNA are composed of optically active (chiral) elements and because they can adopt different types of three-dimensional structures, each type of molecule produces a distinct CD spectra (figure 1). Figure 1. CD spectra of triosephosphate isomerase (helices (H) : 0.52, beta-sheets (S) : 0.14, turns (T) : 0.11, other structure types (O) : 0.23), hen egg lysozyme (H:0.36, S:0.09, T:0.32, O:0.23), myoglobin (H:0.78, S:0.0, T:0.12, O:0.10), and chymotrypsin (H:0.10, S:0.34, T:0.20, O:0.36). Theory Recall that electromagnetic radiation is a complex wave form that can be considered to behave as two simple wave motions at right angles to each other (figure 2). Figure 2. Electromagnetic wave consisting of a sinusoidal oscillations of an electric field and a magnetic field. One of these waves is magnetic (M, green) and the other is electric (E, blue). Electromagnetic waves are generated by oscillating electric or magnetic dipoles and are propagated at the speed of light (c). Since the E- and the M-components are always perpendicular to each other, it is sufficient, in many cases, to consider only the E-component in describing the wave. Although the amplitude of the E-wave oscillates in the zx-plane in the figure above, it could oscillate in any direction perpendicular to the direction of propagation (z). Unpolarized light, the type we get from the sun or a light bulb contains oscillations of the E - components in all directions perpendicular to the direction of propagation. Linearly polarized light results when the direction of the E-component is restricted to a plane perpendicular to the direction of propagation while its magnitude oscillates. Circularly polarized light is another form of polarization - in this case, the magnitude of the oscillation is constant and the direction oscillates (figure 3). Figure 3. Schematic diagram showing the electronic component of linearly polarized light (left) and right-handed circularly polarized light (right). Below each are the electronic component vectors as viewed along the axis of propagation from left of the diagram above. The differential absorption of radiation polarized in two directions as function of frequency is called dichroism. When applied to plane polarized light, this is called linear dichroism; for circularly polarized light, circular dichroism. 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". 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 (histidine, cysteine, tryptophan, tyrosine and phenylalanine) in proteins and the purine and pyrimidine bases in DNA. Specifically, protein secondary and tertiary structures can be monitored by the peptide transitions in the far UV (~190-220 nm) and by the aromatic side chain transitions in the near UV (~270-290 nm), respectively. Other chromophores in the visible region (for coloured proteins) may be valuable for establishing the fidelity of tertiary and quaternary folding. CD spectra can then be analyzed for the different secondary structural types: alpha helix, parallel and antiparallel beta sheet, turn, and other (figure 4). Figure 4. Circular dichroism spectra of "pure" secondary structures. Redrawn from Brahms & Brahms, 1980. Applications CD has an important role in the structural determinants of proteins. However, the effort expended in determining secondary structure elements is usually not worth it because it is somewhat unreliable. The real power of CD is in the analysis of structural and conformational changes in a protein upon some perturbation (salt, pH, temperature). These changes can be quantitated by CD since shifts in conformation involving as few as 10 amino acids may be readily detectable. It can also be efficiently used in comparison of the structure of an engineered protein to the parent protein. For example, it is required that the overall structure of a mutated protein hasn’t changed considerably compared to the wild type protein. Previously, a hydrophobic dyes (for example Nile Red) was used to detect changes, but its use was limited due to the uncertainty of the results. Also, the the changes had to be extremely drastic (a hydrophobic core displayed on the outside of a protein). CD spectroscopy can therefore elucidate whether a change of a single amino acid has caused any slighter perturbations on the overall secondary structure topology. The effect would be similar as in protein denaturation, the studying of which CD spectroscopy fits well, too (figure 5). Figure 5. Protein denaturation – renaturation equilibrium CD can also be used in an empirical manner to determine protein unfolding, ligand or drug binding, etc. CD is rapid and can be used to analyze a number of candidate proteins from which interesting candidates can be selected for more detailed structural analysis like NMR or X-ray crystallography. In advance of obtaining a crystal structure, CD can be useful in conjunction with modelling efforts: it can provide a good test of the secondary structural content of any model produced, and can be used to determine the class of molecule. For crystallography, it may be used as an assay of whether a particular structure will be a reasonable model for molecular replacement.