New Insights in the Interpretation of Tryptophan Fluorescence

Abstract

Origin of tryptophan fluorescence is still up to these days a quiz which is not completely solved. Fluorescence emission properties of tryptophan within proteins are in general considered as the result of fluorophore interaction within its environment. For example, a low fluorescence quantum yield is supposed to be the consequence of an important fluorophore-environment interaction. However, are we sure that the fluorophore has been excited upon light absorption? What if fluorophore excitation did not occur as the result of internal conformation specific to the fluorophore environment? Are we sure that all absorbed energy is used for the excitation process? Fluorescence lifetimes of Trp residues are considered to originate from rotamers or conformers resulting from the rotation of the indole ring within the peptide bonds. However, how can we explain the fact that in most of the proteins, the two lifetimes 0.5 and 3 ns, attributed to the conformers, are also observed for free tryptophan in solution? The present work, performed on free tryptophan and tyrosine in solution and on different proteins, shows that absorption and excitation spectra overlap but their intensities at the different excitation wavelengths are not necessarily equal. Also, we found that fluorescence emission intensities recorded at different excitation wavelengths depend on the intensities at these excitation wavelengths and not on the optical densities. Thus, excitation is not equal to absorption. In our interpretation of the data, we consider that absorbed photons are not necessary used only for the excitation, part of them are used to reorganize fluorophore molecules in a new state (excited structure) and another part is used for the excitation process. A new parameter that characterizes the ratio of the number of emitted photons over the real number of photons used to excite the fluorophore can be defined. We call this parameter, the emission to excitation ratio. Since our results were observed for fluorophores free in solution and present within proteins, structural reorganization does not depend on the protein backbone. Thus, fluorescence lifetimes (0.5 and 3 ns) observed for tryptophan molecules result from the new structures obtained in the excited state. Our theory allows opening a new way in the understanding of the origin of protein fluorescence and fluorescence of aromatic amino acids.

Energy Transfer Studies between Trp Residues of Three Lipocalin Proteins Family, α1-Acid Glycoprotein, (Orosomucoid), β-Lactoglobulin and Porcine Odorant Binding Protein and the Fluorescent Probe, 1-Aminoanthracene (1-AMA)

“…The same results were obtained when interaction between calcofluor white and β-lactoglobulin was studied and where the authors showed that protein emission occurs from Trp 19 residue [33]. Absence of emission of Trp 61 residue of β-lactoglobulin could be explained by the fact that this tryptophan is not excited as the result of its close interaction with disulfide bridge or any other amino acids [34]. At neutral pH, β-lactoglobulin and OBP are dimers.…”

Fluorescence properties of porcine odorant binding protein Trp 16 residue

“…2). The two shortest lifetimes correspond to what we can usually observe for Trp residues in protein or for free tryptophan in solution [11], while the longest lifetime is the result of the interaction of the Trp residue within its environment. The mean fluorescence lifetime (¼ 4.788 ns), calculated from two experiments and measured at 340 nm, was used to measure the rotational correlation of the Trp residues from the Perrin plot.…”

“…The two shortest fluorescence lifetimes (0.8 and 2.7 ns) are in general found in most proteins and for free tryptophan in solution. Thus, these two fluorescence lifetimes emanate from structural rearrangements of Trp residue in the excited state independent of the tryptophan microenvironment [11]. Nevertheless, the three lifetimes increase with the emission wavelengths in the same way, the longest lifetime increases from 4.8 ns at 310 nm to 8 ns at 370 nm and the middle lifetime increases from 1 to 2 ns in the same range of emission wavelength.…”

“…These data are in good agreement with ours (high Trp residue mobility and in contact with both hydrophobic and hydrophilic environments). Also, the pre-exponential factor of each of the three lifetimes varies from one protein to another [11], indicating that its value is sensitive to the Trp residue(s) microenvironment.…”

Characterization of binding mode of imatinib to human α1-acid glycoprotein

“…Excitations were performed both at 280 nm and 295 nm, where emissions reflect the contribution of all aromatic amino acids or only that of Trp residues, respectively [31]. Results showed (Fig.…”