Physical mechanisms of an unconventional green fluorescent protein indicator for chloride

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 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 with a combination of theoretical and experimental investigations. Constant pH molecular dynamics simulations (CpHMD) reveal a coordinated entry of anions into an unconventional binding cavity near the chromophore. Photophysical measurements of the wild-type protein confirm such a possibility and indicate that anion binding tunes the chromophore equilibrium, resulting in a turn-off fluorescence response at acidic pH. Finally, targeted mutagenesis of the anion entry path emphasizes the guiding force of theory for cgreGFP-like indicators and beyond.