Fluorescence in Protein Science


Fluorescence spectroscopy is an optical technique used to study biological systems such as membranes, nucleic acids and proteins, thanks to its high sensitivity and noninvasive nature. In the case of proteins, fluorescence is currently used for analytical detection, to study ligand binding and complex formation and to observe the presence of intermediate species in folding pathways. Resonance energy transfer, lifetime and anisotropy measurements are quantitative applications of fluorescence spectroscopy that allow the characterisation of biological processes in which proteins play fundamental roles, including membrane binding, enzyme catalysis, signalling and the regulation of the function of nucleic acids. The development of new fluorophores, light sources and detectors has opened the possibility to perform such kinds of measurements in vivo, extending the fluorescence technology to cell microscopy.

Key Concepts

  • Protein intrinsic fluorescence arises mainly from aromatic amino acids and some cofactors.
  • The timescale of tryptophan lifetime is the same as protein conformational changes.
  • Fluorescence anisotropy decays provide information on protein dynamics.
  • Fluorescence energy transfer is used to measure distances of the order of protein size.
  • Fluorescence correlation spectroscopy is a valid tool for investigating protein dynamics in vivo.

Keywords: protein fluorescence; fluorescence anisotropy; FRET; protein folding; fluorescence microscopy

Figure 1. (a) Simplified diagram of energy levels for an absorption/re‐emission process. (b) Fluorescence emission spectra of native (blue) and unfolded azurin (in the presence of 6.5 M guanidinium hydrochloride, red) from Pseudomonas aeruginosa, upon excitation at λ = 275 nm).
Figure 2. A cartoon representing a fluorescence anisotropy measurement. Emission is collected at 90° with respect to excitation (in purple) and the two components (parallel, blue; perpendicular, green) separated through polarisers. At excitation time, only those molecules (in purple) with the absorption dipole oriented in the same direction of the excitation beam (i.e. vertical) absorb light (photoselection). Proteins are assumed to be spherical (inset) and characterised by two depolarising effects: the tumbling of the overall molecule (φ1) and the movement of the tryptophan side chain (φ2).
Figure 3. Simulation of a protein unfolding measurement monitored by FRET. The donor is a tyrosine residue (purple) and the acceptor (A) a tryptophan (green). In the folded protein, (a), the donor–acceptor distance is small and the energy transfer is very efficient: under such conditions, no trace of tyrosine emission is detectable in the fluorescence spectrum, which will be probably blue shifted if the tryptophan is partially buried. As the protein unfolds (b and c), the distance increases; the tryptophan signal moves to longer wavelengths and tyrosine emission will appear as a shoulder on the left side of the spectrum (generally around 303 nm).
Figure 4. The diagram summarises 10 principal areas of investigation in protein science in which fluorescence techniques are mostly used.


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Further Reading

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Demchenko AP (1986) Ultraviolet Spectroscopy of Protein. Berlin: Springer‐Verlag.

Lakowicz JR (ed) (2001) Protein fluorescence. In: Topics in Fluorescence Spectroscopy, vol. 6. New York: Kluwer Academic/Plenum Publishers.

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Mei, Giampiero() Fluorescence in Protein Science. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0027584]