Origin of protein fluorescence

The values of the two angles are illustrated in the Ramachandran diagram wiiich defines the limits of conformaUrmal freedom for each peptide bond unit and hence (or the entire polypeptide chain. However, even if limited confcamailons are allowed for each amino acid, the number of possible conformations for the polypeptide Is high. F r example, a 100-residue protein with three confbrmaitons for each amino acid would have 3 possible conformations. Therefore, the number of conformal ion.s that a protein can possess is very important 

A folded protein could have a set of different conformations, thus we have here a first definition of a protein structure the global structure is a coininnation of sub-structures or conformations. The interconversion between them is not too fast. Each conformation is rigid and has a definite specific structure. This model is known as the rotamers modd. 

A fast intercoiveram between differoil sub- iecies induces a more complex system, hi tins case, fluotescence measurements would be a mean value of all the different possible sub-species. 

The main question that is still asked up to now is what is die origin of the tiyptophan fluorescence lifetime in proteins All scientists agree with the fact that when a tryptophan is buried inside the hydrophobic core of a protein, its flucnescence is bloe-shifted compared to the fluocescence observed from a tiyptc ihaD present at the laotein surface. However, the oii ofdie fluotescence Ufetime is still in great debate. 

The rolamers model was used to explain the origin of the biexponential decay of tryptophan free in solution. In polypeptides, lifetime of each retainer is explained as the result of the quenching interactions between the indole and quenching groups in the fluorophore. 

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Albani, J.R. (2007). New insights in the interpretation of tryptophan fluorescence. Origin of the fluorescence lifetime and characterization of a new fluorescence parameter in proteins the emission to excitation ratio. /. of fluorescence, in press. 

Literature contains an important number of papers dealing with proteins fluorescence and espeaally with the origin of the Tryptophan fluorescence. All these studies correlate the origin of the fluorescence to the primary and tertiary structures of the proteins. In peptides and proteins, rotation of the polypeptide chain is observed at the level of the a. carbon atom of each amino acid. This rotation is characterized by two well defined angles of torsion or dihedral < ) and y (Figure 7 ]). 

In this example, it is difficult to assign a specific fluorescence lifetime to a specific Trp residue. Also, the rotamers model can in no way explain the origin of the fluorescence lifetimes. One can consider the fluorescence lifetime as being the result of the Trp residues interaction witli their microenvironments. However, since fluorescence lifetimes do not vary significantly witli die different mutants, one should consider the possibility of having, around the Trp residues, a common identical protein structure responsible of the three measured fluorescence lifetimes. 

Fluorescence intensity decay of the Trp residues ( ex, 300 nm) yields four fluorescence lifetimes, 0.22, 0.44, 2.06 and 4.46 ns. The authors considered the two shortest lifetimes as the result of the excited state quenching of the tryptophan emission by the protein backbone, while the two longest lifetimes are characteristics of the emission of the Trp residues from two different environments (Kung et al. 1998). Tryptophan free in solution emits with two lifetimes, 0.5 and 3.1 ns. In this case, the short lifetime cannot be assigned to the presence of a backbone protein. The two lifetimes are considered to be the result of the different side chain retainers conformations. Thus, a tryptophan in presence of different environments, water and protein matrix, can have the same or identical fluorescence lifetimes. This example shows clearly that it is not obvious to explain the origin of the fluorescence lifetime of tryptophan residues in proteins by the rotamers model. 

In order to understand the origin of the fluorescence of the Trp residues in a protein, we compared the fluorescence parameters of a i-acid glycoprotein prepared in two different ways 1) by a successive combination of ion displacement chromatography, gel filtration and ion exchange chromatography (ai-acid glycoprotein ) and 2) by ammonium sulfate precipitation (ai-acid glycoprotein ). 

In the present two cases, the results described in Table 7.9 indicate that the rotamers model is not adequate to describe the origin of the fluorescence lifetime of the tryptophans in the two proteins. The shortest fluorescence lifetime (35 or 45 ps) found also for tryptophan residues in myoglobin is an indication of the high energy transfer Forster type from tryptophans to heme. We believe that this lifetime is common or almost all hemoproteins where energy transfer between tryptophan(s) and the heme is very important. 

Martin ME, Negri F, Olivucci M (2004) Origin, nature, and fate of the fluorescent state of the green fluorescent protein chromophore at the CASPT2//CASSCF resolution. J Am Chem Soc 126 5452... 

Liu T, Callis PR, Hesp BH, de Groot M, Buma WJ, Broos J (2005) Ionization potentials of fluoroindoles and the origin of nonexponential tryptophan fluorescence decay in proteins. J Am Chem Soc 127(11) 4104-4113... 

Another distinction should be made (independently of the fluorescence aspects) between chemical sensors (also called chemosensors) and biosensors. In the former, the analyte-responsive moiety is of abiotic origin, whereas it is a biological macromolecule (e.g. protein) in the latter. 

In the majority of cases, fluorescent labels and probes, when studied in different liquid solvents, display single-exponential fluorescence decay kinetics. However, when they are bound to proteins, their emission exhibits more complicated, nonexponential character. Thus, two decay components were observed for the complex of 8-anilinonaphthalene-l-sulfonate (1,8-ANS) with phosphorylase(49) as well as for 5-diethylamino-l-naphthalenesulfonic acid (DNS)-labeled dehydrogenases.(50) Three decay components were determined for complexes of 1,8-ANS with low-density lipoproteins.1 51 1 On the basis of only the data on the kinetics of the fluorescence decay, the origin of these multiple decay components (whether they are associated with structural heterogeneity in the ground state or arise due to dynamic processes in the excited state) is difficult to ascertain. 

It should be noted that the dynamics studied by fluorescence methods is the dynamics of relaxation and fluctuations of the electric field. Dipole-orientational processes may be directly related to biological functions of proteins, in particular, charge transfer in biocatalysis and ionic transport. One may postulate that, irrespective of the origin of the charge balance disturbance, the protein molecule responds to these changes in the same way, in accordance with its dynamic properties. If the dynamics of dipolar and charged groups in proteins does play an important role in protein functions, then fluorescence spectroscopy will afford ample opportunities for its direct study. 

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