Ultrafast Structural Evolution and Chromophore Inhomogeneity inside a Green-Fluorescent-Protein-Based Ca²⁺ Biosensor

Abstract

Understanding excited-state structural dynamics of fluorescent-protein-based biosensors for Ca(2+) imaging is crucial for developing new in vivo Ca(2+) indicators and advancing bioimaging. We implemented wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) with a 530 nm Raman pump to uncover the working mechanism of an intensiometric fluorescent-protein biosensor, G-GECO1.1, highlighting the deprotonation process of its embedded chromophore. Besides confirming the dynamic difference of excited-state proton transfer (ESPT) in the Ca(2+)-free/bound protein, we revealed a chromophore two-ring twisting process with time constants of 36/60 ps that competes with ESPT. In contrast with FSRS data collected using the 800 nm Raman pump, the bluer Raman pump enables us to access a subset of reactant population with partially deprotonated character that exhibits an additional ESPT component on the ∼5 ps time scale. These findings provide deep mechanistic insights into the inhomogeneity and subpopulation-specific conformational dynamics of biosensor chromophores, which will guide the rational design of improved biosensors for metal ion imaging.