posted on 2023-12-18, 21:06authored byJasmine
N. Tutol, Whitney S. Y. Ong, Shelby M. Phelps, Weicheng Peng, Helen Goenawan, Sheel C. Dodani
Beyond its role as
the “queen of electrolytes”, 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 the anion-sensitive
fluorescent protein mNeonGreen (mNG). The targeted engineering of
two non-coordinating residues, namely K143 and R195, in the chloride
binding pocket of mNG coupled with an anion walking screening and
selection strategy resulted in the ChlorON sensors: ChlorON-1 (K143W/R195L),
ChlorON-2 (K143R/R195I), and ChlorON-3 (K143R/R195L). In vitro spectroscopy revealed that all three sensors display a robust turn-on
fluorescence response to chloride (20- to 45-fold) across a wide range
of affinities (Kd ≈ 30–285
mM). We further showcase how this unique sensing mechanism can be
exploited to directly image labile chloride transport with spatial
and temporal resolution in a cell model overexpressing the cystic
fibrosis transmembrane conductance regulator. Building from this initial
demonstration, we anticipate that the ChlorON technology will have
broad utility, accelerating the path forward for fundamental and translational
aspects of chloride biology.