Watching an Engineered Calcium Biosensor Glow: Altered Reaction Pathways before Emission.

Biosensors have become an indispensable tool set in life sciences. Among them, fluorescent protein-based biosensors have great biocompatibility and tunable emission properties but their development is largely on trial and error. To facilitate a rational design, we implement tunable femtosecond stimulated Raman spectroscopy, aided by transient absorption and quantum calculations, to elucidate the working mechanisms of a single-site Pro377Arg mutant of an emission ratiometric Ca biosensor based on a green fluorescent protein-calmodulin complex. Comparisons with the parent protein and the Ca-free/bound states unveil more structural inhomogeneity yet an overall faster excited-state proton-transfer (ESPT) reaction inside the Ca-bound biosensor. The correlated photoreactant and photoproduct vibrational modes in the excited state reveal more chromophore twisting and trapping in the Ca-bound state during ESPT and the largely conserved chromophore dynamics in the Ca-free state from parent protein. The uncovered structural dynamics insights throughout an ESPT reaction inside a calcium biosensor provide important design principles in maintaining a hydrophilic, less compact, and more homogeneous environment with directional H-bonding (from the chromophore to surrounding protein residues) via bioengineering methods to improve the ESPT efficiency and quantum yield while maintaining photostability.