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Circular Dichroism Applications:

Protein Structure Analysis Overview

 

Circular dichroism is the foremost technique for investigating protein structure in solution. In the UV and visible wavelength regions, CD spectroscopy detects the electronic transitions of the chromophores in a protein. In the far UV region (170-260nm) the peptide linkages mainly contribute to spectra whereas in the near UV region (240-360nm) the main contributors are the chromophoric-aromatic amino acid side-chains (tryptophan, tyrosine, phenylalanine) and disulphide linkages. In the visible region (350-700nm) other intrinsic or extrinsic chromophores, such as haeme groups or bound chromophoric transition metal ions, are dominant when present.

As a consequence of its connection with electronic transitions, UV and visible wavelength CD spectroscopy is becoming more frequently known as “electronic” CD, so as to differentiate it from the more recent technique of vibrational CD that employs infra-red light.

As a chiroptical technique, CD is inherently sensitive to the chirality of the chromophores and their surrounding environment; achiral molecules, in the absence of any chiral influence, give zero CD (nb. the application of a magnetic field can give magnetic CD effects, these are separate to the existence of chiral molecular geometries).

Proteins are effectively giant, highly chiral structures, both through the stereochemistry of the individual amino acid residues and the inherent macro chirality of the secondary structures, such as “right handed” α-helices, “twisted” β-sheets and barrels, “left-handed” pPII helices, turns, etc.; almost every secondary structural motif of a protein is chiral in some manner. Consequently, proteins are exceptionally good samples for CD spectroscopy. In addition, anything bound to the protein, such as a putative drug or ligand, will be invariably placed in a chiral environment and may likewise start contributing to the CD spectrum, even if not chiral itself when in isolation, so further extending the capabilities of CD to binding studies.

Importantly, CD spectroscopy simply requires a sample solution to be in a medium (solvent) that is transparent in the wavelength region of interest, of which water is exceptionally good, being inherently transparent from near infra-red through to far UV wavelengths. As such, proteins can be typically studied in aqueous media approaching that of their natural environments – a huge advantage for assuring relevance.

Of course, other components of a solution, such as buffers, have to be likewise carefully chosen so as not to obscure any protein spectrum due to their absorbance; many suitable choices are readily available and often employed in biomolecular studies in general. This is less of an issue for the near UV and visible regions but can usually be readily overcome in the more demanding far UV region. Beyond aqueous environments, it is also possible to study proteins in a wide variety of organic solvents, to probe structural flexibility and stability or mimic membranes.

Most commercial CD spectrometers allow the simultaneous acquisition of an isotropic (unpolarised) absorption spectrum at the same time as the CD spectrum; it is strongly advisable to avail oneself of this opportunity. Both spectra should be inspected and interpreted alongside each other, as much can be gained from this. Not least, indications of artefacts and spurious features will be easier to spot, such as scattering from particulate matter (perhaps precipitated protein), chromophoric contaminants or oxygen absorptions due to insufficient nitrogen flushing in the instrument

    

  

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References

For further literature see our Literature page