Directed Evolution of a Genetically Encoded Indicator for Chloride

Inarguably,
the green fluorescent protein (GFP) family is an exemplary
model for protein engineering, accessing a range of unparalleled functions
and utility in biology. The first variant to recognize and provide
an optical output of chloride in living cells was serendipitously
uncovered more than 25 years ago. Since then, researchers have actively
expanded the potential of GFP indicators for chloride through site-directed
and combinatorial site-saturation mutagenesis, along with chimeragenesis.
However, to date, the power of directed evolution has yet to be unleashed.
As a proof-of-concept, here, we use random mutagenesis paired with
anion walking to engineer a chloride-insensitive fluorescent protein
named OFPxm into a functional indicator named ChlorOFF. The sampled
mutational landscape unveils an evolutionary convergent solution at
one position in the anion binding pocket and nine other mutations
across eight positions, of which only one has been previously linked
to chloride sensing potential in the GFP family.