About GFP...

Chromophore

The technical revolution resulting from the discovery of GFP relates to a particular property of the chromophore that is responsible for its fluorescence2. This chromophore is formed spontaneously from a tri-peptide motif in the primary structure of GFP: the maturation only requires oxygen and does not depend on the presence of enzymes or other auxiliary factors. Hence, fluorescence is turned on in every organism where GFP is expressed.

Moreover, the barrel-like structure of these proteins effectively shield the chromophore from the external environment5. GFP and its related variants thereby provide universal genetic tags that can be used to visualize a virtually unlimited number of spatio-temporal processes in virtually all living systems2.

Fluorescence excitation (full-line curve) and emission (dashed curve) spectra of native GFP from Aequorea victoria.

Excited state dynamics6

In the wild-type Aequorea green fluorescent protein, the chromophore exists as an equilibrating mixture of the neutral phenol (absorbance lmax= 397 nm, extinction coefficient = 25000 M-1cm-1) and anionic phenolate (absorbance lmax= 475 nm, extinction coefficient = 9500 M-1cm-1)7. Regardless of whether excitation is at 397 nm or 475 nm, the fluorescence emission occurs from the anionic phenolate species (fluorescence lmax= 504 nm) with a quantum yield of 0.797. Excitation of the neutral phenol species results in the fast (tens of picoseconds) excited state proton transfer (ESPT)8 of the phenol proton to an internal hydrogen bond network9 . Variants of Aequorea green fluorescent protein with the ground state equilibrium shifted to either the phenol or phenolate species are particularly useful for fluorescence imaging applications. A blue fluorescent variant (fluorescence lmax= 456 nm) with both the ground and excited state equilibriums shifted toward the phenol has been reported10.

Variations in chromophore structure6

In terms of natural variations of fluorescent protein chromophores, researchers have now discovered at least 5 chromophores with chemically distinct conjugated systems.

Fluorescent protein chromophore structures known to occur in nature are shown below. All structures are shown as the Z stereoisomer (more common than the E stereoisomer).

A) The Aequorea green fluorescent protein chromophore. B) The Discosoma red fluorescent protein chromophore.
C) The Zoanthus yellow fluorescent protein chromophore. D) The Anemonia sulcata "kindling" fluorescent protein chromophore.
E) Trachyphyllia geoffroyi "Kaede" red fluorescent protein chromophore.

Each of these chemical structures is associated with a range of fluorescence hues, following the general trend that more extended conjugation produces longer wavelength fluorescence. For example, the structures shown in A, B and C are associated with greenish, reddish and yellowish hues, respectively. The exact excitation and emission wavelengths for a particular chromophore structure also depends strongly on the protein microenvironment that surrounds the chromophore. For example, naturally occurring cyan fluorescent proteins from Anemonia majano, Discosoma striata, and Clavularia sp have Aequorea green fluorescent protein-type chromophores (A), but substantially blue-shifted emission due to electrostatic interactions with the surrounding protein microenvironment.

Another type of variation in chromophore structure is the stereochemistry (F). In principle, every type of fluorescent protein chromophore could exist as either the Z or E stereoisomer depending on the steric constraints of the protein microenvironment and/or prior illumination. For example, the Discosoma red fluorescent protein chromophore is the Z stereoisomer shown in B, while a non-fluorescent chromoprotein from Montipora efflorescens and a far-red fluorescent protein from Entacmaea quadricolor exist as the E stereoisomer of this same chromophore structure. Photoinduced isomerizations between Z and E isomers in the same protein have also been shown to occur in some cases11.

In addition to inducing interconversion of stereoisomers, illumination can also result in photochemical reactions that change the covalent structure of the chromophore. For example, Trachyphyllia geoffroyi "Kaede" red fluorescent protein initially forms a typical Aequorea green fluorescent chromophore. Subsequent illumination with ultraviolet light results in an elimination reaction across the C-C bond of the adjacent histidine side chain, thus cleaving the main chain of the polypeptide and producing the red fluorescent chromophore shown in E.

Outstanding properties2

From the perspective of its usefulness in the biosciences, some aspects of GFP function stand out. Among these are:

  1. the brightness of the molecule, defined as the extinction coefficient at the maximum of the excitation spectrum multiplied by the quantum yield (quantum yield is the probability of luminescence occurring in given conditions, expressed by the ratio of the number of photons that are emitted by the luminescing species to the number of photons that are absorbed5);
  2. the photo-stability of the molecule, i.e. the average number of photons that the chromophore emits before the fluorescence is lost due to chemical events emanating from the first singlet excited state and leading to photo-decomposition;
  3. the existence of GFP-like molecules with different excitation and emission spectra throughout the whole visible region;
  4. rapid and efficient folding of the molecule in the intracellular context;
  5. rapid maturation of the chromophore subsequent to protein folding;
  6. monomeric configuration of GFP-like proteins, to facilitate their fusing with proteins of interest.

Great brightness (1.) and photo-stability (2.) are determinants of the signal-to-noise ratio of GFP-derived intracellular signals. Variable excitation and emission properties (3.) will, in particular, allow for the monitoring of events when two GFP molecules, where the emission spectrum of one (the donor) matches the excitation spectrum of the other (the acceptor), come close to each other. The principle is usually referred to as Fluorescence (or Förster) Resonance Energy Transfer (FRET). FRET can be used to estimate inter-chromophoric distances smaller than about 100 Å, and the method has been generalized to involve energy transfer between three, rather than two, GFP chromophores.

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