Coupling a Live Cell Directed Evolution Assay with Coevolutionary Landscapes to Engineer an Improved Fluorescent Rhodopsin Chloride Sensor

Our
understanding of chloride in biology has been accelerated through
the application of fluorescent protein-based sensors in living cells.
These sensors can be generated and diversified to have a range of
properties using laboratory-guided evolution. Recently, we established
that the fluorescent proton-pumping rhodopsin wtGR
from Gloeobacter violaceus can be converted into
a fluorescent sensor for chloride. To unlock this non-natural function,
a single point mutation at the Schiff counterion position (D121V)
was introduced into wtGR fused to cyan fluorescent
protein (CFP) resulting in GR1-CFP. Here, we have integrated coevolutionary
analysis with directed evolution to understand how the rhodopsin sequence
space can be explored and engineered to improve this starting point.
We first show how evolutionary couplings are predictive of functional
sites in the rhodopsin family and how a fitness metric based on a
sequence can be used to quantify the known proton-pumping activities
of GR-CFP variants. Then, we couple this ability to predict potential
functional outcomes with a screening and selection assay in live Escherichia coli to reduce the mutational search space of
five residues along the proton-pumping pathway in GR1-CFP. This iterative
selection process results in GR2-CFP with four additional mutations:
E132K, A84K, T125C, and V245I. Finally, bulk and single fluorescence
measurements in live E. coli reveal that GR2-CFP
is a reversible, ratiometric fluorescent sensor for extracellular
chloride with an improved dynamic range. We anticipate that our framework
will be applicable to other systems, providing a more efficient methodology
to engineer fluorescent protein-based sensors with desired properties.