Tuning calcium biosensors with a single-site mutation: structural dynamics insights from femtosecond Raman...

Fluorescent protein biosensors are popular reporters for biological processes and life sciences, but their fundamental working mechanisms remain unclear. To characterize the functional fluorescence events on their native timescales, we implemented wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) to shed light on a blue-green emission-ratiometric fluorescent protein based Ca²⁺ biosensor with a single Pro377Arg mutation. The transient Raman modes of the embedded chromophore from ca. 1000-1650 cm^-1 exhibit characteristic intensity and frequency dynamics which infer the underlying atomic motions and photochemical reaction stages. Our experimental study reveals the hidden structural inhomogeneity of the protein local environment upon Ca²⁺ binding with the mutated arginine residue trapping multiple chromophore subpopulations, which manifest distinct time constants of ∼16 and 90 ps for excited state proton transfer (ESPT) following 400 nm photoexcitation. The altered ESPT reaction pathways and emission properties of the Ca²⁺ biosensor represent the foundational step of rationally designing advanced fluorescent protein biosensors to tune their functionalities by site-specifically altering the local environment (e.g., the active site) of the embedded chromophore.