The Fluorescent Protein Barrel as a Programmable Electrostatic Device Reveals Distinct Light-Sensitive Fluorescence States of the Chromophore.

Genetically encoded voltage indicators (GEVIs) enable optical readout of membrane potential by coupling conformational changes in a voltage-sensing domain to fluorescence transitions within a cytoplasmic fluorescent protein (FP) domain. Although the FP β-barrel largely shields the chromophore, the mechanisms by which voltage-dependent perturbations are transduced into optical signals remain incompletely understood. Here, we show that the FP β-barrel functions as a programmable electrostatic environment that governs voltage-dependent fluorescence transitions. Systematic manipulation of β-strand electrostatics reveals that directional polar offsets partition the fluorescence response into separable components whose relative contributions depend on internal residue identity near the chromophore. Disruption of steady-state fluorescence further resolves these components into distinct photophysical processes with differential light sensitivity. Comparison of responses under excitation conditions that preferentially engage protonated or anionic chromophore states demonstrates that chromophore state influences how these transitions are accessed. Extending electrostatic perturbations to a β-strand discontinuity in the β-barrel interfacing with the chromophore proton network further modulates response amplitude and kinetics, while in some cases producing nonuniform spatial responses in the cell. More broadly, these results recast the FP β-barrel not as a passive shield but as an electrostatically tunable determinant of chromophore photophysics-suggesting, beyond voltage sensing, a general strategy for engineering fluorescent proteins with tailored optical properties.