A Twisted Chromophore that Powers a Fluorescent Protein Chloride Sensor

Chromophore nonplanarity and flexibility have long been argued to govern the fluorescence efficiency of fluorescent proteins (FPs), yet their relative importance has been elusive. Herein, we tackle this question by investigating two FP-based chloride (Cl–) sensors, ChlorON1 and 3, using ultrafast spectroscopy and theoretical calculations. We find that fluorescence enhancement of the chloride-bound ChlorON3 stems from a more twisted chromophore than ChlorON1. This counterintuitive finding indicates that chromophore planarity is not, but conformational rigidity is, the decisive factor for high fluorescence efficiency.