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Circular Dichroism/Optical Rotary Dispersion



Plane polarized light is made up of a left-handed (L) and a right-handed component (R) which are equal in magnitude. Circular Dichroism (CD) refers to the difference in absorption of these two components by a sample. Only chiral chromophores will produce a CD signal which can occur for the following reasons:

  • An intrinsically chiral chromophore such as a carbon atom bonded to four different ligands or a disulphide bond with chirality because of dihedral angles in its C-S-S-C chain of atoms.
  • Chromophores covalently bonded to a chiral center.
  • Chromophores placed in an asymmetric environment due to the 3D structure of the molecule

If the sample does not absorb or equally absorbs both the L and R components, then the resulting radiation should be polarized in the original plane. However, if circular dichroism occurs, than the resulting radiation will posses an elliptical polarization. CD instruments generally measure this elliptical polarization (ellipticity) in terms of degrees (θ):
θ = tan-1 (b/a)
b = minor axis of the resultant ellipse
a = major axis of the resultant ellipse

Optical rotatory dispersion (ORD) is a technique used to study the chirality of a biomolecule and consists of passing a beam of linearly polarized light through the sample. If there is any degree of chirality in the sample, then the light will be rotated as a function of wavelength and from this rotation, one can determine the chirality (left- or right-handed) of the molecule.

Applications

  • Protein/Peptide conformational studies
  • Thermal and chemical denaturation studies
  • Structure determination

Instrument Info

J815 (Jasco, Installed Feb. 2010). The instrument includes a Peltier temperature control device for thermal denaturations. Optical rotatory dispersion (ORD) capability.

Sample Requirements

  • 0.1cm sample cell: 200uL of sample
  • 0.2cm sample cell: 300uL of sample
  • Larger cells are available on request and you are welcome to bring your own quartz cells
  • For Far UV Measurements (190nm-250nm):
    • Peptides: 0.1mg/mL (Typical)
    • Nucleic Acids: 0.5 OD (Typical)
  • For Near UV (250nm-350nm)
    • Peptides: 1mg/mL

Training

Training consists of a brief walkthrough of the instrument and its operating procedures with core facility personnel. This is required before any experiments can be performed.

To get a basic feel for the procedure of the experiment, it is recommended that you review this tutorial before coming in to use the instrument.

Conversions & Prediction Programs

  • See the Biophysics Core Handbook for conversions and other handy information used in CD studies.
  • K2D M.A. Andrade, P. Chacón, J.J. Merelo, and F. Morán, European Molecular Biology Laboratory, Heidelberg, Germany.

The K2D method uses a self-organising neural network to extract the secondary structure features present in the data from a set of circular dichroism spectra ranging from 200 nm to 241 nm.

The network is trained using a number of reference spectra and the information is stored in a matrix for recall. k2d then uses this pre-calculated data to predict the secondary structures present based on your CD data. The run does not take more than a few seconds and provides an estimation of the percentages of helix, sheet and random structure of your protein. k2d also gives the probable error in the estimation, based on its training procedure.

  • ANTHEPROT (ANalyse THE PROTeins) is the result of about 10 years of biocomputing activity of a group of the Institute of Biology and Chemistry of Proteins. The main idea was to integrate into a single package most of the methods designed for protein sequence analysis (1,2,3,4). This package contains a number of analysis routines that predict secondary structure based on CD data.
  • DICROPROT (DICHROism of PROTeins) is from the same group as ANTHEPROT and integrates most of the methods designed for the estimation of protein sequence secondary structure derivation from circular dichroism experiments into a single package. The major goal in making DICROPROT is to help the user to manage the spectra acquired with Jobin-Yvon apparatus.
  • CD 222:208 ratio 1.10 => Coiled coil

   CD 222:208 ratio 0.90 = >  Helix

   Molar Ellipticity Online Conversion