Illuminating Photochemistry of an Excitation Ratiometric Fluorescent Protein Calcium Biosensor

Fluorescent protein (FP)-based biosensors have become an important and promising tool to track metal ion movement inside living systems. Their working principles after light irradiation, however, remain elusive. To facilitate the rational design of biosensors, we dissect the fluorescence modulation mechanism of a newly developed excitation ratiometric green FP-based Ca²⁺ biosensor, GEX-GECO1, using femtosecond stimulated Raman spectroscopy (FSRS) in the electronic excited state. Upon 400 nm photoexcitation, characteristic vibrational marker bands at ∼1180 and 1300 cm^–1 show concomitant decay and rise dynamics, probing the progression of an ultrafast excited state proton transfer (ESPT) reaction. The Ca²⁺-bound biosensor exhibits two distinct populations that undergo ESPT with ∼6 and 80 ps time constants, in contrast to one dominant population with a 25 ps time constant in the Ca²⁺-free biosensor. This result is supported by key structural constraints from molecular dynamics simulations with and without Ca²⁺. The blueshift of the ∼1265 cm^–1 C–O stretch mode unravels the vibrational cooling dynamics of the protonated chromophore regardless of Ca²⁺ binding events. This unique line of inquiry reveals the essential structural dynamics basis of fluorescence modulation inside an excitation ratiometric protein biosensor, correlating the uncovered chromophore structural heterogeneity with different H-bonding configurations and intrinsic proton transfer rate in the photoexcited state