Chapter 84 - Ultrafast dynamics in the excited state of wild-type Green Fluorescent Protein

Abstract

The photoactive protein, green fluorescent protein (GFP), from the pacific jellyfish Aequoria Victoria is prototypical for bioluminescence from P-barrel tertiary structures. This structural motif serves to protect the chromophore from the aqueous surroundings and hence, to prevent solvent- assisted quenching after photo-excitation. Furthermore, the P-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 Forster-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 corresponding to absorptive resonances centered around 400 nm and 477 nm, which can interconvert by means of proton transfer. 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. This chapter presents 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.