The ChlorON Series: Turn-on fluorescent protein sensors for imaging labile chloride in living cells
Beyond its role as the queen electrolyte, chloride can also serve as an allosteric regulator or even a signaling ion. To illuminate this essential anion across such a spectrum of biological processes, researchers have relied on fluorescence imaging with genetically encoded sensors. In large part, these have been derived from the green fluorescent protein found in the jellyfish Aequorea victoria. However, a standalone sensor with a turn-on intensiometric response at physiological pH has yet to be reported. Here, we address this technology gap by building on our discovery of mNeonGreen (mNG) derived from lanYFP found in the cephalochordate Branchiostoma lanceolatum. Targeted engineering of two non-coordinating residues in the chloride binding pocket of mNG unlocks the ChlorON series. In vitro spectroscopy reveals that the binding of chloride tunes the chromophore environment to give rise to the turn-on response. We further showcase how this unique sensing mechanism can be exploited for directly imaging labile chloride in living cells with spatial and temporal resolution, accelerating the path forward for fundamental and translational aspects of chloride biology. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/503807v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@36cc4org.highwire.dtl.DTLVardef@d9dccborg.highwire.dtl.DTLVardef@12ec0fforg.highwire.dtl.DTLVardef@80c9db_HPS_FORMAT_FIGEXP M_FIG C_FIG