Phenomena Index Green Fluorescent Protein

Intrinsic Fluorescence of Proteins and Peptides

Proteins contain three aromatic amino acid residues (tryptophan, tyrosine, phenylalanine) which may contribute to their intrinsic fluorescence. Cofactors such as FMN, FAD, NAD and porphyrins also exhibit fluorescence. A special case of fluorescence occurs in Green Fluorescent Protein where the fluorophore originates from an internal serine-tyrosine-glycine sequence which is post-translationally modified to a 4-(p-hydroxybenzylidene)- imidazolidin-5-one structure. Changes in intrinsic fluorescence can be used to monitor structural changes in a protein.

The fluorescence of a folded protein is a mixture of the fluorescence from individual aromatic residues. Protein fluorescence is generally excited at 280 nm or at longer wavelengths, usually at 295 nm. Most of the emissions are due to excitation of tryptophan residues, with a few emissions due to tyrosine and phenylalanine.

The three residues have distinct absorption and emission wavelengths. They differ greatly in their quantum yields and lifetimes. Due to these differences and to resonance energy transfer from proximal phenylalanine to tyrosine and from tyrosine to tryptophan, the fluorescence spectrum of a protein containing the three residues usually resembles that of tryptophan.

The table summarizes the fluorescence characteristics of the three aromatic residues:

Lifetime is shown in nanoseconds, wavelength in nanometers, along with the molar absorptivity and quatum yield of the fluorophore. Generally, lifetimes are short and quantum yields are low for all three residues.

Tryptophan (shown as free acid) has much stronger fluorescence and higher quantum yield than the other two aromatic amino acids. The intensity, quantum yield, and wavelength of maximum fluorescence emission of tryptophane is very solvent dependent. The fluorescence spectrum shifts to shorter wavelength and the intensity of the fluorescence increases as the polarity of the solvent surrounding the tryptophane residue decreases. Tryptophan residues which are buried in the hydrophobic core of proteins can have spectra which are shifted by 10 to 20 nm compared to tryptophans on the surface of the protein. Tryptophan fluorescence can be quenched by neighbouring protonated acidic groups such as Asp or Glu.

Tyrosine, like tryptophan, has strong absorption bands at 280 nm, and when excited by light at this wavelength, has characteristic emission profile. Tyrosine is a weaker emitter than tryptophan, but it may still contribute significantly to protein fluorescence because it usually present in larger numbers. The fluorescence from tyrosine can be easily quenched by nearby tryptophan residues because of energy transfer effects. Also, tyrosine can undergo an excited state ionization which may result in the loss of the proton on the aromatic hydroxyl group that leads to quenching of tyrosine fluorescence.

Phenylalanine with only a benzene ring and a methylene group is weakly fluorescent. The experimental sensitivity (the product of quantum yield and molar absorbtivity maximum) is especially low for this residue. Phenylalanine fluorescence is observed only in the absence of both tyrosine and tryptophane. The simple structure of phenylalanine may preeminently demonstrate the effect of structure on fluorescence. Adding a hydroxyl group, as in tyrosine, causes a 20 fold increase in fluorescence. If an indole ring is added as in tryptophan, the relative fluorescence increases to 200 times that of phenylalanine.

Rotamers of Tryptophan

Tryptophan in peptides and proteins frequently exhibits biexponential decay kinetics, which may suggest the presence of conformers in equilibrium in the folded structure.

In human serum albumin (left), which is a protein with only one tryptophan residue (shown in yellow), a long (T₁=3.14 nsec) and a short (T₂ = 0.51 nsec) lifetime is observed. By measuring the fluorescence spectra at different times during the decay, the short and long component peaks at 335 and 250 nm, respectively.

The biexponencial decay can be attributed to a single electronic transition of tryptophan, which may exist as different conformational isomers in the protein. This is illustrated in the figure below using Newman projection, ie. looking at the alpha carbon along the alpha-beta carbon axis:

Because of steric effects between the side chain of tryptophan and the polypeptide backbone, all rotamers are not equally probable. The rotamer with the greatest population and the lifetime of 3.1 nsec is A in the figure, where the quenching group nearest to the indole is the small amino group. Rotamer B is somewhat less probable, as the larger carbonyl group is the closest one to the indole. If not quenched, its lifetime maybe in the same order, though. The least likely rotamer is C, with both amino and carbonyl group close to the indole. This rotamer may have the short lifetime of 0.51 nsec. The presence of different tryptophan rotamers has been independently confirmed by NMR spectroscopy. Thus the uniqueness of three-dimensional structure appears as a somewhat a relative term, at least in solution. The structure may be better described by the presence of conformers in equilibrium.

The presence of rotamers, however, does not fully explain the multiexponential decay in single tryptophane proteins. As the indole ring assumes different positions in a polypeptide chain, slightly differnt position of neighbouring quenching groups may also result in multiexponential decay.

The Effect of Folding on Intrinsic Fluorescence

The quantum yields for all three aromatic amino acids decrase when they are incorporated into a polypeptide chain. The table compares the fluorescent characteristics of the free amino acids and internal amino acid residues. It also contains data to show the sensitivity of fluorescence of these residues to the polarity of their environment.

^*DMSO = dimethyl sulfoxide, an organic solvent for amino acids and peptides, with dielectric constant of 36. The dielectric constant of H₂0 is 80.

The fluorescence of the aromatic residues varies in somewhat unpredictable manner in various proteins. Comparing to the unfolded state, the quantum yield may be either incresed or decreased by the folding. Accordingly, a folded protein can have either greater or less fluorescence than the unfolded form. The intensity of fluorescence is not very informative in itself. The magnitude of intensity, however, can serve as a probe of perturbations of the folded state. The wavelenght of the emitted light is a better indication of the environment of the fluorophore. Tryptophan residues that are exposed to water, have maximal fluorescence at a wavelength of about 340-350 nm, whereas totally buried residues fluoresce at about 330 nm.

Emission maxima, lifetimes and links to entries in the PDBsum database are listed listed below for some single tryptophane proteins that have been studied extensively by fluorescence spectroscopy:

Emission maximum is in nm, lifetime is in nsec.

Extrinsic Fluorophores

Covalent Fluorescent Protein Labels

Proteins can be covalently labeled with various fluorophores, thus producing fluorescent protein conjugates. The emission from these attached tags is called extrinsic fluorescence. Tagging a protein with fluorescent labels is an important and valuable tool for studying structure and microenvironment. Commonly used labeling reagents fall into certain classes on the basis of the type of group on the protein with which they react. Each class of reagent then can be subdivided according to the reactive group on the reagent. The major classes are:

Labels attaching to primary amino groups:

• Arylsulfonyl halydes
• Isocyanates and isothiocyanates
• Nitrobenzoxadialoles
• N-succinimides and anhydrides

Thiol group labels:

• Haloacetamides
• Maleimides
• Aziridines
• Disulfides
• Bimanes

Many other miscallenous labels have proven to be useful. For more details, see References.

Specific labeling of proteins usually involves nontrivial chemistry. A labeling ratio of 1:1 does not guarantee a unique label site. Inadvertent labeling may limit the usefulness of some external fluorophores.

Noncovalent Fluorescent Ligands

Fluorescent molecules which bind to proteins have frequently been used to monitor structure and conformation. Unlike covalently attached fluorophores, these non-covalently bound ligands are in equilibrium with unbound ligans. Ligands which have been used to probe protein binding sites often have the property of fluorescing significantly only when bound. The change in fluorescence upon binding is due to the difference in excited state dipole reorientation in the binding site as compared with that in aqueous solution. The large solvent effects are due to a large increase in dipole moment in the excited state, followed by solvent relaxation.

In most cases where noncovalently bound ligand is used as fluorescent probe, the ligand is anionic. A large number of proteins can bind such anionic ligands at their active site. Only a few cationic ligands have been useful to probe active sites. Most natural substrates are anionic at physiological pH. An example is Auramine O which binds to the active site of alcohol dehydrogenase. Trypsin whose substrates have the positively charged side chains of basic amino acids (lysine and arginine), can bind the positively charged ligand proflavin.

Phenomena Index Green Fluorescent Protein

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