Abstract
Fluorescent proteins bearing an intrinsic tripeptide chromophore exhibit diverse, tunable photophysical features that are exceptional for biosensing applications. However, atomic-level details of these sensing mechanisms are obscured experimentally, particularly as protein motions, including the chromophore, cannot be divined from the structure alone. Molecular dynamics (MD) simulations can bridge this gap, providing a landscape of global and local protein motions with resolution to key amino acids connected to function and potential for engineering. In this study, we uncover that the green fluorescent protein from the jellyfish Clytia gregaria (cgreGFP) is sensitive to anions, including chloride, bromide, iodide, and nitrate, with a combination of theoretical and experimental investigations. Constant pH molecular dynamics (CpHMD) simulations reveal a coordinated entry of all four anions into an unconventional binding cavity near the chromophore. Photophysical measurements of the wild-type protein confirm this behavior and demonstrate that anion binding tunes the chromophore equilibrium, resulting in a turn-off fluorescence response at acidic pH with the affinity trend iodide > nitrate > bromide > chloride. Finally, targeted mutagenesis of the anion entry pathway emphasizes the guiding force of theory to understand cgreGFP-like indicators and beyond.