Circular dichroism for protein characterisation


During my PhD project, I was working on a bacterial protein involved in iron transport. The secondary and tertiary information about this protein was limited and function was not well known at that time. In order to shed more light on this, I was looking for various techniques and came across ‘Circular Dichroism’ [CD], a widely used technique for analysing the secondary structure content of the protein of interest in solution. Circular dichroism measures the difference in the absorption of left and right circularly polarised light by chiral molecules. It is also a great technique to study the protein-ligand interactions.

The secondary structure composition is associated with characteristic spectra based on the absorption due to the peptide bond in the far ultraviolet [UV] CD region (180-260 nm) whereas tertiary structure features are influenced by the spectrum in the near UV region (260-320 nm) with absorption being due to aromatic residues [2]. In this category, synchrotron radiation circular dichroism spectroscopy [3] [SRCD] also became popular which provided more structural information with the available options for measuring the spectra to lower wavelengths, with improved signal to noise ratio levels.

I came across an article in Nature Scientific Reports indicating more advancement in this area by the introduction of High-throughput SRCD [HT-SRCD] using multi-well plates [1] which was interesting. The paper describes the features provided by Beamline B23 of Diamond Light Source, Oxford, UK for setting up the HT-SRCD using multi-well plates. It also describes examples for high-throughput measurements carried out using multi-well systems. In short, this HT-SRCD could be a potential resource for studying the protein folding, conformational changes in protein structure induced by ligands, buffers and other components as well as secondary structure determination on a high-throughput scale.

Blog written by Mohan Rajasekaran

  1. Hussain, R., Javorfi, T., Rudd, T. R., and Siligardi, G. (2016) High-throughput SRCD using multi-well plates and its applications. Scientific reports 6, 38028
  2. Kelly, S. M., Jess, T. J., and Price, N. C. (2005) How to study proteins by circular dichroism. Biochimica et Biophysica Acta (BBA) – Proteins and Proteomics 1751, 119-139
  3. Wallace, B. A., and Janes, R. W. (2010) Synchrotron radiation circular dichroism (SRCD) spectroscopy: an enhanced method for examining protein conformations and protein interactions. Biochemical Society transactions 38, 861-873

 

 

 

 

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