Turning the Fluorescent Protein Barrel into a Programmable Electrostatic Device Reveals Differential Bleaching States of the Chromophore

Genetically encoded voltage indicators (GEVIs) based on fluorescent proteins (FPs) report membrane potential changes through voltage-driven conformational rearrangements of a voltage-sensing domain that perturb the electrostatic and hydrogen-bonding environment of a fused FP chromophore. While the chromophore is largely protected from the external environment by the FP {beta}-can, previous results suggest that this structure can also act as a programmable electrostatic grid that influences chromophore flexibility and fluorescence transition states. Here, we show that varying the external polar offset of {beta}-sheet residues flanking a chromophore-proximal position systematically reshapes fluorescence transitions, altering response polarity and kinetics. Importantly, the influence of these external residues depends strongly on the internal sidechain chemistry, revealing reciprocal coupling between {beta}-sheet electrostatics and chromophore-proximal residues. We further show that the functional effects of glutamine and asparagine substitutions at this position are strongly context-dependent and can be tuned by appropriate external polar offsets, consistent with electrostatic control of internal sidechain orientation and expanding the molecular switch repertoire. Disrupting steady-state fluorescence further revealed that the secondary component of the voltage-dependent fluorescence transition is preferentially light-sensitive and can be selectively diminished by repeated stimulation, indicating that it arises from a photophysically distinct process separable from the primary response. In addition, altering the chromophore protonation equilibrium (e.g., T65S) substantially changes response kinetics, indicating that chromophore state influences how {beta}-can electrostatics couple to fluorescence transitions. Together, these results define general programming rules by which the electrostatic grid of the FP {beta}-barrel can be tuned to control chromophore optical properties both at rest and during perturbation of the steady state.