The technique

The plane polarization of light is a phenomenon familiar to anyone who has polaroid sunglasses or a glare-reducing polarization filter for a camera lens. Polarized light arises from the summation of two components, left- and right-circularly polarized light (LCPL and RCPL). If these components are of equal intensity and in phase, plane-polarized light results, If not in phase or of different intensity, their interaction generates elliptically polarized light. Molecules, such as proteins, that have chiral centres (e.g. the alpha-carbon atoms, disulfide bonds, or aromatic side chains) which absorb one of the circularly polarised light components slightly more than the other, resulting an elliptical polarisation of the transmitted light that is measured by the spectrometer. This optical activity is associated with peaks of the absorption spectrum and hence peptide bonds show circular dichroism (CD) activity in the region from 250 nm to 178 nm (the lowest wavelength that can be recorded on most CD spectrometers) and the aromatic side chains around 300-260 nm. Some protein ligands or prosthetic groups show CD activity at higher wavelengths. Nucleic acids also have CD spectra at wavelengths where they absorb light.

Applications

  1. Determine secondary structure of a protein. From the CD spectrum one can calculate regions of the protein that adopt alpha-helical, parallel and antiparallel beta-sheet, beta-turn, and "random coil" secondary structures.
  2. Determine if the structure of an expressed protein has folded correctly, especially if native protein is available for comparison.
  3. Follow the unfolding of a protein, i.e. one can follow the denaturation and renaturation of proteins.
  4. Determine changes in the environment of the aromatic amino acid side chains (another measure of folding or unfolding) as well as of interactions with other molecules, cas measured around 280 nm.

CD spectroscopy is the fastes way of checking protein folding.  It takes 10-20 minutes to record a full spect

Facilities available

Jasco J-710 Circular Dichroism Spectrometer (Room 441A Molecular Biosciences Building) with thermostated cuvette holder.
 

Sample and set-up requirements

Protein concentration should be 11 mg/ml with little or no buffer/salst (as spectra must be recorded from 250 nm to 180 nm or lower).  Belwo 200nm, oxygen buffers and salts (including NaCl and NaN3) absorb strongly and the signal-to-noise ratio therefore falls abruptly:  this is partly offset by averaging multiple scans).

Cuvettes with different pathlengths are available (0.1mm, 1mm, 10 mm) depending on sample requirements.  Between ~35 and 200 ul samples are needed depending on cuvette used.  Optical activity in aromatic region i saround 10-fold waker and thus its measurement requires a higher protein concentration or longer pathlength (typically 1 mm to 1 cm).

Most of the protein sample can be recovered from cells if needed.

Contact

Dr Amanda Nouwens
a.nouwens@uq.edu.au
3346 9490

Charges for use

For non-SCMB UQ staff and students:  $40 per session

For researchers from other non-profit organisations:  $80 per session

UQ users have priority of access.

 

  

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