Attend this on-demand webinar presented by Prof Jihad René Albani (Université des Sciences et Technologies de Lille, France) and through experimental examples you will discover the origin of fluorescence lifetimes of L-tryptophan free in solution and in peptides and proteins.
We present for the first time straight forwarded experiments describing origin of fluorescence lifetimes of L-tryptophan free in solution and in peptides and proteins. Two lifetimes (0.5 and 2.5 ns) were observed when L-Trp is dissolved in pure hydrophilic solvent (water) or pure hydrophobic solvent (chloroform and carbon tetrachloride). Populations of both lifetimes (short and longest one) are the same in both solvents (0.1 and 0.9, respectively), indicating that emission is occurring from two specific populations formed each by the tryptophan backbone with its electronic distribution in the excited state. Excitation spectra of L-Trp in chloroform and in water are different; therefore, emission does not occur from conformers existing in the ground state.
In ethanol, a third lifetime is observed and is the result of interactions occurring between the tryptophan and hydrophobic / hydrophilic chemical functions of ethanol.
In proteins, three lifetimes are also observed for Tryptophan residue. Two lifetimes are identical to the two observed in pure water and / or in ethanol. Thus, these two lifetimes are inherent to the tryptophan itself, independently of its surrounding environment. In proteins, the third lifetime originates from the interactions that are occurring between tryptophan residues and neighboring amino acids. Fluorescence decay studies of different tripeptides having a tryptophan in second position show that the best analysis is obtained with two fluorescence lifetimes. Consequently, this result seems to exclude the possibility that peptide bond induces the third fluorescence lifetime.
Indole dissolved in water and / or in ethanol emits with two fluorescence lifetimes that are completely different from those observed for L-Trp. Absence of the third lifetime in ethanol demonstrates that indole behaves differently when compared to tryptophan. Thus, it seems not adequate to attribute fluorescence lifetime or fluorescence properties of tryptophan to indole ring and to compare tryptophan fluorescence properties in proteins to molecules having close structures such as NATA which fluoresces with one lifetime.
Free in solution or when in proteins, tryptopohan lifetimes and pre-exponential values are independent of the excitation energy, this means that emitting sub-structures or populations are not arbitrary but pre-existing, pre-defined ones and they are revealed after excitation occurs.
In other words, description of molecules with one structure, as it is done today, is oversimplified.
- Tryptophan emission occurs from pre-existing substructures in the excited state and revealed after excitation.
- Emission of tryptophan in proteins occurs with three fluorescence lifetimes. Two lifetimes are inherent to the tryptophan itself and are independent of its environment. The third lifetime is the result of the interaction between tryptophan residue and neighbouring amino acids.
- Description of a molecule with one structure, as it is done today, is oversimplified.
Jihad René Albani (Université des Sciences et Technologies de Lille, France) research consists mainly on the study of the relation that exists between structure, dynamics and function of proteins. He uses fluorescence spectroscopy as the main technique of investigation. The fluorescence lifetimes allow to get an idea on the heterogeneity of the studied system, the red-edge excitation spectra method helps the study of the dynamics of the fluorophore microenvironment, the fluorescence anisotropy reveals the dynamics of the fluorophore itself and the quenching of the fluorescence allows to calculate the affinity constant of a complex or to measure the diffusion constant within the system studied. He has published 40+ scientific papers and 5 reviews on invitation on different proteins including 20+ papers on α1-acid glycoprotein.