Institute for Lasers, Life and Biophotonics, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands
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Phys. Chem. Chem. Phys., 2012,14, 8852-8858
05 Mar 2012,
15 Mar 2012
First published online
15 Mar 2012
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 H2O, 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.
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