Unraveling Ultrafast Photoinduced Proton Transfer Dynamics in a Fluorescent Protein Biosensor for Ca2+ Imaging

Imaging Ca2+ dynamics in living systems holds great potential to advance neuroscience and cellular biology. G-GECO1.1 is an intensiometric fluorescent protein Ca2+ biosensor with a Thr-Tyr-Gly chromophore. The protonated chromophore emits green upon photoexcitation via excited-state proton transfer (ESPT). Upon Ca2+ binding, a significant population of the chromophores becomes deprotonated. It remains elusive how the chromophore structurally evolves prior to and during ESPT, and how it is affected by Ca2+. We use femtosecond stimulated Raman spectroscopy to dissect ESPT in both the Ca2+-free and bound states. The protein chromophores exhibit a sub-200fs vibrational frequency shift due to coherent small-scale proton motions. After wavepackets move out of the Franck-Condon region, ESPT gets faster in the Ca2+-bound protein, indicative of the formation of a more hydrophilic environment. These results reveal the governing structure-function relationship of Ca2+-sensing protein biosensors.