Lichtinduzierte Primärprozesse in reversibel photoschaltbaren fluoreszierenden Proteinen: Zeitaufgelöste Spektroskopie von Padron0.9 und rsFastLime

Abstract

Within this project, the photodynamics of the reversibly switchable fluorescent proteins Padron0.9 and rsFastLime after photoexcitation were investigated. Cis/trans-isomerization of the chromophore is an essential element of the reversible switching between a fluorescent ON- and a non-fluorescent OFF-state observed on the proteins Padron0.9 and rsFastLime. Both isomers establish an equilibrium between the chromophor’s protonated and deprotonated form. The different chromophore species were isolated and selectively excited to elucidate the main reaction pathways by ultrafast pump-probespectroscopy. Intermediate states and the corresponding spectra were identified by extensive spectral and kinetic modelling of the obtained transient data. For both proteins, the strongly fluorescent ON-state corresponds with the deprotonated cis-species. After excitation, it decays monoexponentially with a lifetime of 1.16 ns and 0.9 ns for Padron0.9 and rsFastLime, respectively. The deprotonated trans-species of Padron0.9 decays within 1 ps, dominated by internal conversion into a hot ground state. About one third relaxes into a more stable excited state with a lifetime of 7 ps. The excited protonated cisrsFastLime has a lifetime of 8 ps, mostly due to internal conversion. About 15 % relax into a more stable exited state with a lifetime of 215 ps. Excitation of protonated cis-Padron0.9 is followed by a fast exited state proton transfer kH = 2.0 ps^-1 to an exited cis-intermediate, identified by a strong H/D-isotope effect. This intermediate has a lifetime of 37 ps and relaxes to a corresponding intermediate ground state and the exited fluorescent ONstate with equal probability. The intermediate ground state is subsequently protonated within 2 ps, as shown by pump-dump-probe spectroscopy. In addition, the disturbed ground state protonation equilibrium is reestablished within 45 ps. Exited state dynamics of protonated trans-rsFastLime are dominated by internal conversion to the ground state within 2.1 ps. One third of the excited molecules relax to a intermediate excited state with a lifetime of 14 ps and subsequently to a more stable exited state with a life time of 94 ps with IV subsequent conversion to a cis-groundstate. For protonated cis-rsFastLime, no evidence for an exited state proton transfer was found. Quantum yields for the photoinduced switching processes found for Padron0.9 are 0.0003 (cis to trans at 500 nm) and 02 (trans to cis at 400 nm). The yields are too low to accurately identify the respective products. Neither rsFastLime protonated isomer’s spectrum can be distinguished, so product formation may occur but cannot be assigned. For Padron0.9, the excited state proton transfer is proposed to be the first step on the inefficient isomerization path trans to cis. Regardless of the quantum yields for isomerization, it was shown that the different ground state protonation equilibrium for Padron0.9 and rsFastLime suffice to explain the inverse switching characteristics of these structually very similar proteins.