Ultrafast dynamics in the excited state of green fluorescent protein (wt) studied by frequency-resolved femtosecond pump-probe spectroscopy

Abstract

The time and frequency resolved optical response of wild-type green fluorescent protein (wt-GFP) has been measured at room temperature following 30 fs, 400 nm photo-excitation. In the wavelength range covering the stationary fluorescence spectrum of the protein, the stimulated emission rises on a time scale of roughly 20 ps due to excited-state proton-transfer (ESPT). The rise can be described phenomenologically by a sum of two exponentials. A long-time isosbestic behavior on the blue edge of the stationary emission implies a barrier for ESPT which is significantly larger than thermal excitations. In addition, an instantaneous component to the stimulated emission appears within the time resolution of our experiment. This observation is indicative of nonvertical cross-well transitions that prepare the proton-transferred configuration of the excited state directly from the equilibrium geometry of the ground-state neutral species during photo-excitation. Finally, transient absorptions around 500 nm and 650 nm can be observed, which are attributed to transitions from different protonated forms of the excited-state of GFP to higher lying electronic configurations, S n . The entire optical response of GFP is quantitatively simulated using a dynamic model that includes: (i) an energy-dependent rate coefficient for ESPT, (ii) intra-and intermolecular transfer of excess vibrational energy (IVR and VET), and (iii) an additional non-radiative decay pathway for the initially prepared Franck-Condon state leading to internal conversion via motion along a torsional coordinate. In particular, the nonexponential nature of the ESPT originates from overlapping time scales of reactive and non-reactive elementary processes following optical excitation.

Uncovering the hidden ground state of green fluorescent protein

“…The difference spectra of A* and I* agree with those reported earlier (9) and resemble the raw time-resolved spectra at delays of 400 fs and 22 ps of Fig. 2, respectively.…”

“…It was postulated that the molecular ground state of the chromophore in this anionic form acts as an intermediate in the GFP photocycle (2,(5)(6)(7)(8). However, with time-resolved spectroscopy, only the excited states A* and I* of GFP could be detected (9), and the molecular nature and dynamics of any ground-state intermediate remained elusive.…”

Concerted Asynchronous Proton Transfer in H-Bonding Relay Model: An Implication of Green Fluorescent Protein

“…The calculated value deviates from the experimentally estimated barrier (2.8 kcal/mol). 26 There are several reasons for this discrepancy. First, it should originate from the full relaxation considered here, which may lead larger barrier.…”

Solvation shift of a conical intersection in clusters of excited tetrakis(dimethyl amino)ethylene with ammonia and acetonitrile molecules

“…The aim is to unravel by which mechanism, the passage of the wavepacket in the CI region is affected by the presence of a medium surrounding the TDMAE molecule. The issue is of importance considering that the CI concept is now routinely invoked to rationalize the dynamics of molecules or of molecular groups that are embedded in solvents [7,8] or proteins [9,10]. This issue is part of a more general concern that has been reviewed recently: the role of cluster solvation in reaction dynamics [11,12].…”