Summary

The primary events which give rise to luminescence from protein environments have recently raised considerable attention [1] and [2]. An elucidation of the underlying photophysical and photochemical elementary processes including their time scales will help establish fundamental principles involved in photon emission from protein structures. Such information is highly valuable in tailoring optical bio-fluorophores with spectrochemical properties that are optimally adapted for applications such as protein/membrane/DNA interactions, gene expression, or non-invasive protein localization. The photoactive protein, green fluorescent protein (GFP), from the pacific jellyfish Aequoria Victoria is prototypical for bioluminescence from β-barrel tertiary structures. This structural motif serves to effectively protect the chromophore (p-hydroxybenzylidene- imidazolidinone) from the aqueous surroundings and hence, to prevent solvent- assisted quenching after photo-excitation. Furthermore, the β-barrel interior provides an extended network of hydrogen-bonds in which the chromophore is embedded and which proves to be essential for a primary excited state proton transfer (ESPT) followed by the characteristic green emission. Temperature-dependent dynamic fluorescence spectroscopy have led to a Förster-type kinetic model in which both the ground and excited electronic states of the GFP chromophore are characterized by two distinct conformations, A and B correspondding to absorptive resonances centered around 400 nm and 477 nm, which can interconvert by means of proton transfer [1], [2] and [3]. In the excited state, proton transfer from A* occurs on a time scale of several picoseconds via barrier crossing to an intermediate, I*, which is responsible for the characteristic green emission. Presumably, I* constitutes an environmentally unrelaxed configuration of the proton-transferred form, B*, of the excited state. Here, we present complementary femtosecond time- and frequency-resolved pump-probe, fluorescence upconversion (FlUp) and time correlated single photon counting (TCSPC) experiments to quantify the elementary dynamics accompanying ESPT in wt-GFP.