Circular dichroism
A spectrum from about 260 down to 190 nm (or lower, on synchrotron or nitrogen-purged instruments) that reports backbone conformation. It is the basis of secondary-structure estimation and the quickest check that a protein is folded at all.
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Far-UV
A spectrum from about 260 down to 190 nm (or lower, on synchrotron or nitrogen-purged instruments) that reports backbone conformation. It is the basis of secondary-structure estimation and the quickest check that a protein is folded at all.
How it differs from the standard method
- Needs about 0.1–0.2 mg/mL in a 1 mm cell, or 1 mg/mL in a 0.1 mm cell, so that total absorbance stays near 1 at 190 nm.
- Highly sensitive to buffer: use 10 mM phosphate with NaF or sulfate for ionic strength, and avoid chloride, Tris, imidazole, DTT and detergents with aromatic groups.
- The 190–200 nm region carries most of the information that separates β-sheet from coil; data that stop at 200 nm give a much less reliable estimate.
- Results depend on an accurate concentration, since every fraction is computed from mean residue ellipticity.
1Left and right, absorbed differently
Circularly polarised light comes in two hands. A chiral molecule absorbs one hand slightly more than the other, and circular dichroism (CD) is that difference, ΔA = A(left) − A(right), measured as a function of wavelength.
2Turning millidegrees into structure
Raw ellipticity depends on how much protein is in the beam, so it is converted to mean residue ellipticity: [θ] = θ × MRW / (10 × l × c), with the mean residue weight MRW in Da, the path length l in cm and the concentration c in mg/mL. Every secondary-structure estimate is downstream of that division.
0.1–0.2 mg/mL, 1 mm
Total absorbance near 1 at 190 nm keeps the detector in range down to the bands that separate sheet from coil.
≈ 1 mg/mL, 10 mm
Aromatic bands are weak; A280 around 1 in a 1 cm cell gives a fingerprint with good signal-to-noise.
Phosphate and fluoride
Chloride, Tris, imidazole, DTT and many detergents absorb in the far UV. Use dilute phosphate and NaF or Na₂SO₄ for salt.
222 nm, ≤ 1 °C/min
Follow the helix band while heating slowly in a stoppered cell, then cool and check the signal comes back.
3How CD measurements fail
The photomultiplier's high-tension (HT) voltage rises automatically as less light reaches it. Where the sample and buffer together absorb too much, the HT climbs past the range in which the instrument responds linearly (on most instruments somewhere around 600–700 V), the noise explodes and the CD values become meaningless, but they still appear on the screen.
4Thermal melts
Following the 222 nm signal while the temperature rises gives an unfolding curve. Fitted to a two-state model with sloping native and unfolded baselines, it yields the midpoint Tm and a van 't Hoff enthalpy from the steepness of the transition.
- Ellipticity (θ)
- The instrument's unit for circular dichroism, in millidegrees; θ(mdeg) ≈ 32 980 × ΔA.
- Mean residue ellipticity
- Ellipticity normalised per peptide bond, in deg cm² dmol⁻¹; independent of concentration and path, and the only form in which spectra can be compared.
- HT voltage
- The photomultiplier's high-tension voltage, which rises as less light reaches the detector. Data above the instrument's limit are not trustworthy.
- Absorption flattening
- Loss of CD amplitude when the chromophores are concentrated in particles, as in aggregates, membranes or vesicles.
Key terms
Practise these as flashcards →The 180–250 nm region, dominated by the peptide backbone; reports secondary structure.
The 250–320 nm region, from aromatic side chains and disulfides in a fixed environment; reports tertiary structure.
Protein mass divided by the number of peptide bonds (residues − 1), typically 110–115 Da.
A collection of proteins of known structure whose CD spectra are used to deconvolute a new spectrum, as in CONTIN, SELCON, CDSSTR or BeStSel.
Normalised root-mean-square deviation between a spectrum and its fit; values above about 0.1 suggest the fit cannot be trusted.
Melting temperature: the midpoint of a thermal unfolding transition, where half the protein is unfolded.
The unfolding enthalpy implied by the steepness of a two-state transition; it equals the calorimetric enthalpy only for a true two-state process.
A compact state with much native secondary structure but loose side-chain packing: far-UV close to native, near-UV signal largely lost.
When to use it
- Checking that a purified recombinant protein, a mutant or a new construct is folded, and comparing it with the wild type, using little material and no labelling.
- Estimating secondary-structure content, or following a change in it with ligand binding, pH, temperature, membranes or a co-solvent.
- Measuring thermal or chemical stability by following one wavelength through an unfolding transition, for example to compare mutants or buffer conditions.
- Distinguishing a native protein from a molten globule by recording both far- and near-UV spectra.
Compared with alternatives
Far higher throughput for Tm and ligand-induced stabilisation in plates, but no structural information; prefer it for screening buffers or compounds.
Measures the unfolding enthalpy and ΔCp directly as heat, and can show multiple domains; prefer it when thermodynamic parameters must be model-free.
Tracks tryptophan environment with less protein and is the usual readout for chemical denaturation; it reports local tertiary changes rather than secondary structure.
Gives the actual structure at atomic resolution; CD gives a global average in solution in minutes, and is often used to check a crystallised construct behaves the same in solution.
Residue-level structure and dynamics in solution for smaller proteins, at far greater cost in sample and time.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
A spectrum from about 260 down to 190 nm (or lower, on synchrotron or nitrogen-purged instruments) that reports backbone conformation. It is the basis of secondary-structure estimation and the quickest check that a protein is folded at all.
- Needs about 0.1–0.2 mg/mL in a 1 mm cell, or 1 mg/mL in a 0.1 mm cell, so that total absorbance stays near 1 at 190 nm.
- Highly sensitive to buffer: use 10 mM phosphate with NaF or sulfate for ionic strength, and avoid chloride, Tris, imidazole, DTT and detergents with aromatic groups.
- The 190–200 nm region carries most of the information that separates β-sheet from coil; data that stop at 200 nm give a much less reliable estimate.
- Results depend on an accurate concentration, since every fraction is computed from mean residue ellipticity.