New Red-Emitting Chloride-Sensitive Fluorescent Protein with Biological Uses

A new chloride-sensitive red fluorescent protein derived from Entacmaea quadricolor is described. We found that mBeRFP exhibited moderate sensitivity to chloride and, via site-directed mutagenesis (S94V and R205Y), we increased the chloride affinity by more than an order of magnitude (k d = 106 ± 6 mM) at physiological pH. In addition, cis–trans isomerization of the chromophore produces a dual emission band with different chloride sensitivities, which allowed us to develop a ratiometric methodology to measure intracellular chloride concentrations.