Circular Dichroism (CD)

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CD at the CMI

Circular Dichroism (CD) Spectroscopy is used to determine the optical isomerism and secondary structure of molecules. Circular dichroism (measured in molar ellipticity) is the difference in absorption of left-handed and right-handed circularly polarized light and can be observed in optically active molecules with chiral centers. Proteins have many chiral centers. CD spectra in the Far-UV region (185 – 250 nm) can be used to determine protein secondary structure. Characteristic peaks for Thermal stability (Tm) can be measured by following changes in molar ellipticity with increasing temperature.

CD Sample Data

The CMI has a Jasco J-815 CD Spectropolarimeter with a Peltier temperature controller and single cuvette holder.

CMI Jasco J-815 CD Getting Started Guide, guide to experimental design and standard Far-UV CD protocols.

 

Protein Circular Dichroism Data Bank, a database of CD data

Dichroweb, an online circular dichroism analysis suite

  

The J-815 User Guide is available at the instrument, but not electronically.

  • High Quality Quartz cuvette
    • Far-UV measurements (protein secondary structure):  1 mm pathlength cuvette (eg. Hellma 110-1-40, style 110-QS, available from Sigma-Aldrich)
    • Near-UV measurements:  1 cm pathlength cuvette (eg. Hellma 100-10-40, style 100-QS, available from Sigma-Aldrich)
  • your sample
  • buffer blank

Assay Buffers

Buffer selection is critical for accurate CD measurements. Solvent absorbance can severely interfere with the CD signal. Many commonly used buffers and additives absorb in the far UV region used for CD measurements.

  • The ideal CD experiment is performed in a buffer:
    • in which your protein is well behaved and soluble
    • with no buffer absorbance through the range of the CD spectrum.
  • Always take a scan of your buffer blank to determine whether absorbance interferes with your region of analysis.
    • Ensure that the buffer blank is well matched to the final dilution of your protein (even trace amounts of some solvents will interfere with the CD measurements).
  • Phosphate buffers with little-to-no NaCl are recommended. 10 mM phosphate buffer is ideal.
  • Ideal aqueous solutions contain as little chloride as possible.
    • NaCl and Tris buffers are not recommended but can be tolerated at low concentrations.
    • If salt is required, SO42- or F- are preferred counter ions, as they are transparent in the far UV. Potassium fluoride may be a good substitute for chloride-containing salts.
    • Chloride ions will result in a loss of signal below about 200±5 nm, which affects secondary structure estimation, but generally will not block detection of the alpha-helical peaks (208 nm and 222 nm) or the beta-sheet peak (218 nm).
  • DTT, ß-ME, or EDTA can be present at low concentrations (1 mM).
  • Some detergents are fairly transparent in the far UV (e.g. Chaps and octylglucoside). Triton detergents should be avoided, as they can oxidize readily and form UV-absorbing materials.
  • DMSO, formamides, and imidazole absorb strongly in the far UV region, and thus should be avoided in a CD experiment.

Samples

  • An accurate protein concentration is required for all CD experiments.
    • Over-estimating or under-estimating the protein concentration interferes with signal
    • Algorithms that estimate secondary structure depend on correct protein concentrations
  • The amount of sample required for a CD experiment depends on the size, cuvette pathlength and type of measurement being performed.
  • 1 mm pathlength cuvettes hold 300 µl and a 1 cm cuvette holds 3 ml.
  • Required protein concentration is inversely proportional to the cuvette pathlength, and thus a 1 mm path cuvette requires 10x the concentration of a 1 cm cuvette.
  • Recommended concentrations for Far-UV measurements of protein secondary structure are:
    • 0.2 mg/mL in 1 mm path cuvette
    • 0.02 mg/mL in 10 mm patch cuvette
    • Mol/L=115/(MW*7000)*10/pathlength(mm).
    • For alpha-helical proteins you may need ½ this concentration. For beta-sheet proteins, you may need double the concentration. If possible, make a concentrated stock solution of your protein (at least 2X) and dilute as needed.
  • Protein aggregates can interfere with the CD signal.
    • Assess protein heterogeneity via light scattering.
    • Purify protein samples with soluble aggregates by size-exclusion chromatography.
  • Samples can be recovered from the cell. This is not recommended for thermal melts, unless you know your thermal denaturation is reversible.