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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 Tryptophan Tyrosine Phenylalanine 2.6 3.6 6.4 Absorption Fluorescence Wavelength Absorptivity Wavelength Quantum 280 5,600 348 0.20 274 1,400 303 0.14 257 200 282 . 0.04

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

14 nsec) and a short (T2 = 0. In human serum albumin (left). like tryptophan. Also. a long (T1=3. By measuring the fluorescence spectra at different . as in tyrosine. Tyrosine is a weaker emitter than tryptophan. the relative fluorescence increases to 200 times that of phenylalanine. causes a 20 fold increase in fluorescence. has strong absorption bands at 280 nm. has characteristic emission profile. Tyrosine. If an indole ring is added as in 10 to 20 nm compared to tryptophans on the surface of the protein. The fluorescence from tyrosine can be easily quenched by nearby tryptophan residues because of energy transfer effects. The experimental sensitivity (the product of quantum yield and molar absorbtivity maximum) is especially low for this residue. which may suggest the presence of conformers in equilibrium in the folded structure. Phenylalanine fluorescence is observed only in the absence of both tyrosine and tryptophane. 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. but it may still contribute significantly to protein fluorescence because it usually present in larger numbers. Phenylalanine with only a benzene ring and a methylene group is weakly fluorescent.51 nsec) lifetime is observed. Tryptophan fluorescence can be quenched by neighbouring protonated acidic groups such as Asp or Glu. Adding a hydroxyl group. which is a protein with only one tryptophan residue (shown in yellow). Rotamers of Tryptophan Tryptophan in peptides and proteins frequently exhibits biexponential decay kinetics. The simple structure of phenylalanine may preeminently demonstrate the effect of structure on fluorescence. and when excited by light at this wavelength.

The presence of different tryptophan rotamers has been independently confirmed by NMR spectroscopy. 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. This is illustrated in the figure below using Newman projection. where the quenching group nearest to the indole is the small amino group. however. ie. This rotamer may have the short lifetime of 0. as the larger carbonyl group is the closest one to the indole. its lifetime maybe in the same order. Thus the uniqueness of three-dimensional structure appears as a somewhat a relative term. If not quenched.times during the decay. which may exist as different conformational isomers in the protein. The least likely rotamer is C. The rotamer with the greatest population and the lifetime of 3. the short and long component peaks at 335 and 250 nm. with both amino and carbonyl group close to the indole.1 nsec is A in the figure. does not fully explain the multiexponential decay in single tryptophane proteins. at least in solution. The presence of rotamers. respectively. It also contains data to show . though. all rotamers are not equally probable. As the indole ring assumes different positions in a polypeptide chain.51 nsec. The table compares the fluorescent characteristics of the free amino acids and internal amino acid residues. The structure may be better described by the presence of conformers in equilibrium. The biexponencial decay can be attributed to a single electronic transition of tryptophan. 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. Rotamer B is somewhat less probable.

Tryptophan residues that are exposed to water. Accordingly.0 1bm0 Nuclease 334 5.006 Tyrosine DMSO 306 0. can serve as a probe of perturbations of the folded state. an organic solvent for amino acids and peptides. 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 Lifetime PDBsum Link Azurin 308 4.02 Solvent * DMSO = dimethyl sulfoxide. the quantum yield may be either incresed or decreased by the folding. a folded protein can have either greater or less fluorescence than the unfolded form.0 1nuc Monellin 342 2. The dielectric constant of H20 is 80.21 --Tryptophan DMSO 340 0.0 1azu RNase T1 324 3.the sensitivity of fluorescence of these residues to the polarity of their environment. The magnitude of intensity. The intensity of fluorescence is not very informative in itself.19 333. lifetime is in nsec. Emission maxima. Amino Acid Polypeptide Emission Quantum Emission Quantum * Phenylalanine DMSO 282 0. whereas totally buried residues fluoresce at about 330 nm. however.8 1gcn Emission maximum is in nm.02 284 0. with dielectric constant of 36.6 1mol Glucagon 352 2. The wavelenght of the emitted light is a better indication of the environment of the fluorophore.5 1bvi HSA 342 6. The fluorescence of the aromatic residues varies in somewhat unpredictable manner in various proteins.27 309 0.06 Tyrosine H20 303 0.81 333 0.67 Tryptophan H20 340 0. have maximal fluorescence at a wavelength of about 340-350 nm. Extrinsic Fluorophores Covalent Fluorescent Protein Labels . 0. Comparing to the unfolded state.

The emission from these attached tags is called extrinsic fluorescence. Commonly used labeling reagents fall into certain classes on the basis of the type of group on the protein with which they react. see References. Ligands which have been used to probe protein binding sites often have the property of fluorescing significantly only when bound. The large solvent effects are due to a large increase in dipole moment in the excited state. thus producing fluorescent protein conjugates. the ligand is anionic. 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. Tagging a protein with fluorescent labels is an important and valuable tool for studying structure and microenvironment. 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. followed by solvent relaxation. In most cases where noncovalently bound ligand is used as fluorescent probe. these noncovalently bound ligands are in equilibrium with unbound ligans. For more details. Noncovalent Fluorescent Ligands Fluorescent molecules which bind to proteins have frequently been used to monitor structure and conformation. Each class of reagent then can be subdivided according to the reactive group on the reagent. Most . Specific labeling of proteins usually involves nontrivial chemistry. Inadvertent labeling may limit the usefulness of some external fluorophores. 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. A labeling ratio of 1:1 does not guarantee a unique label site.Proteins can be covalently labeled with various fluorophores. Unlike covalently attached fluorophores.

can bind the positively charged ligand proflavin. 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).natural substrates are anionic at physiological pH. Phenomena Index Green Fluorescent Protein .