Excited state proton transfer in strongly enhanced GFP (sGFP2)Electronic supplementary information (ESI) available: (1) Photoconversion in sGFP2. (2) Comparison of kinetics of sGFP2 and GFPuv. (3) Model based data analysis. See DOI: 10.1039/c2cp40694b

Proton transfer is an elementary process in biology. Green fluorescent protein (GFP) has served as an important model system to elucidate the mechanistic details of this reaction, because in GFP proton transfer can be induced by light absorption. We have used pump-dump-probe spectroscopy to study how proton transfer through the 'proton-wire' around the chromophore is affected by a combination of mutations in a modern GFP variety (sGFP2). The results indicate that in H 2 O, after absorption of a photon, a proton is transferred (A* → I*) in 5 ps, and back-transferred from a ground state intermediate (I → A) in 0.3 ns, similar to time constants found with GFPuv, although sGFP2 shows less heterogeneous proton transfer. This suggests that the mutations left the proton-transfer largely unchanged, indicating the robustness of the proton-wire. We used pump-dump-probe spectroscopy in combination with target analysis to probe suitability of the sGFP2 fluorophore for super-resolution microscopy. Ground- and excited-state proton transfer was studied by pump-dump-probe spectroscopy combined with target analysis, and super-resolution microscopy application is discussed.