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‐200 fs 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.