Intrinsic fluorescence
Track folding and binding through tryptophan fluorescence.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
1A built-in reporter
Most proteins carry their own fluorophores: tryptophan, tyrosine and, very weakly, phenylalanine. Tryptophan dominates.
2Choosing what to excite and what to read
Tryptophan only
Tyrosine absorbs very little beyond about 290 nm, so 295 nm excitation gives a clean tryptophan signal.
Tryptophan and tyrosine
More light absorbed and more signal, but tyrosine emission near 303 nm and energy transfer to tryptophan change on unfolding too.
Where the peak is
Tracks exposure to water: about 330 nm buried, about 350 nm exposed.
The intensity-weighted mean
Moves nearly in proportion to the fraction unfolded; better for fitting than the raw peak position.
Every emission spectrum also contains light that is not the protein. Water scatters a small fraction of the excitation light with a fixed energy loss (the Raman band, about 3400 cm⁻¹ below the excitation: near 310 nm when exciting at 280 nm and near 328 nm at 295 nm).
3Measuring stability by chemical denaturation
The standard use of intrinsic fluorescence in stability work is a denaturant titration. Separate samples of protein are prepared across a range of urea or guanidinium chloride (GdmCl) concentrations, left to reach equilibrium, and read.
- Cm
- The denaturant concentration at which half the protein is unfolded; ΔG = 0 there.
- m-value
- The slope of ΔG against denaturant concentration, in kJ/mol/M; proportional to the change in accessible surface area on unfolding.
- ΔG(H₂O)
- The free energy of unfolding extrapolated to zero denaturant, equal to m·Cm in the linear model.
- Hysteresis
- A gap between the unfolding and refolding curves: the samples have not reached equilibrium, or unfolding is not reversible.
Key terms
Practise these as flashcards →The wavelength of maximum emission; for tryptophan about 308–355 nm depending on how exposed and how polar its surroundings are.
Movement of the emission to longer wavelengths, as when a buried tryptophan becomes exposed to water on unfolding.
The intensity-weighted mean emission wavelength; a smoother, more nearly linear measure than the peak position.
Inelastic scattering from water at a fixed energy shift from the excitation; a sharp band that moves with the excitation wavelength, not with the protein.
Loss of fluorescence through contact with, or energy transfer to, nearby groups such as disulfides, protonated histidines or the peptide backbone.
Fitting denaturation data with ΔG falling linearly with denaturant concentration, and extrapolating to zero denaturant.
Loss of measured intensity because the sample absorbs the excitation or emitted light; keep the absorbance below about 0.1.
A strong chemical denaturant, typically used up to 6–8 M; ionic, so it also screens electrostatic interactions.
When to use it
- Measuring the conformational stability of a protein at a defined temperature, ΔG(H₂O) and m, by chemical denaturation with little material.
- Comparing the stability of mutants, or of one protein in different buffers, where ΔΔG between variants is the quantity of interest.
- Following ligand binding that changes a tryptophan's environment, as a titration of fluorescence quenching or enhancement.
- Watching folding and unfolding kinetics in a stopped-flow instrument, where tryptophan fluorescence is the usual probe.
Compared with alternatives
Reports secondary structure directly and is the usual second probe of a denaturation curve; if CD and fluorescence give the same curve, the transition is likely two-state.
nanoDSF uses the same tryptophan signal in capillaries for thermal melts; prefer it for screening Tm across many conditions.
Gives ΔH and ΔCp of thermal unfolding without an optical probe; combined with ΔG from chemical denaturation it defines the full stability curve.
Measures binding of a labelled ligand through its rotation rather than a change in tryptophan environment; prefer it when the binding site has no tryptophan nearby.
The instrument side: gain, slits and inner-filter corrections; read it before moving a cuvette assay into plates.