Search


Partenaires

CNRS
 
UDS

Unité mixte CNRS - UDS
(UMR 7504)




IPCMS
23 rue du Loess
BP 43
67034 STRASBOURG Cedex 2
France

Tel. +33 3 88 10 70 00
Fax +33 3 88 10 72 50


Home page > Highlight > Archives of the highlights > Formation dynamics and nature of tryptophan’s primary photoproduct in aqueous solution

Formation dynamics and nature of tryptophan’s primary photoproduct in aqueous solution

Version imprimable de cet article Version imprimable

Like other amino-acids tryptophan (Trp) is a building block of proteins. Spectroscopically though, it is very specific, since it is the brightest fluorophore among natural amino acids and its (UV) fluorescence emission spectrum and yield are strongly affected by its molecular environment. Therefore Trp fluorescence spectroscopy has long been established as a local probe for protein structure. For instance, the fluorescence spectrum strongly shifts depending on whether a given Trp residue is exposed to water or buried inside the protein. This has been used to address the issue of protein folding/unfolding. Alternatively, the fluorescence lifetime (or quenching) strongly depends on whether the electronic system of Trp is interacting with another one in a so-called pi-stacking interaction, an important mechanism for biomolecular recognition.

However, the precise understanding of Trp photophysics remains far from trivial. Even when isolated in water, Trp fluorescence decay is multiexponential due to a conformation-dependent quenching mechanism debated over several decades. We have performed UV-VIS Transient Absorption (TA) spectroscopy in order to identify the formation dynamics and nature of the photoproduct resulting from S1 quenching of Trp in water.

We discovered a new, spectral signature which is rising with the same multiexponential time dependence as what was previously observed for the fluorescence decay (see figure). Hence we conclude it is the absorption spectrum of the transient photoproduct resulting from excited state quenching. Like what was observed for fluorescence decay, the TA dynamics is sensitive to pH, and the time evolutions (photoproductrise) exactly match what was reported by fluorescence spectroscopy for the excitedstate decay. In addition we compare to the TA of Trp when it is incorporated in a protein, where we know that an excited-state electron transfer is responsible for the fast fluorescence quenching. In this case, we do not observe the above photoproduct signature. Based on our observations and on previous works, we conclude that the quenching mechanism for Trp in water is due to a proton transfer rather than an electron transfer, thus concluding a 30 year-old debate. We also reveal the absorption spectrum and lifetime of this early photoproduct. Quantum chemistry modeling is now required in order to get deeper insight into the complex photophysics of Trp.

Figure: A) Transient absorption change of Trp in Water, at pH=7, upon UV excitation at 266 nm. At early times (0.06 ns) we observe the excited-state absorption from the S1 state of Trp, with a band around 325 nm and a weaker one around 450nm. While time evolves up to 6 ns, this spectral signature decays (clearly seen around 325 nm and 500 to 600 nm) to gives rise to a new spectrum characterized by two pronounced absorption bands at 350 nm and 425 nm attributed to the photoproduct.

Figure: B) In water at neutral pH, Trp is in a zwitterionic form. Following UV light excitation, the dominant S1 quenching mechanism is an intramolecular, excited-state proton transfer occurring on a few ns time scale.

Formation dynamics and nature of tryptophan’s primary photoproduct in aqueous solution, J. Léonard, D. Sharma, B. Szafarowicz, K. Torgasin and S. Haacke, Phys. Chem. Chem. Phys. 2010, doi: 10.1039/c0cp00615g.
Contact: Jeremie Leonard