Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation
- Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation
- Joshua S. Grimley; Li Li; Weina Wang; 레이웬; Lorena S. Beese; HommeW. Hellinga; 조지어거스틴
- Issue Date
- Journal of neuroscience
- VOL 33, NO 41, 16297-16309
- We describe an engineered fluorescent optogenetic sensor, SuperClomeleon, that robustly detects inhibitory synaptic activity in single,
cultured mouse neurons by reporting intracellular chloride changes produced by exogenous GABA or inhibitory synaptic activity. Using
a cell-free protein engineering automation methodology that bypasses gene cloning, we iteratively constructed, produced, and assayed
hundreds of mutations in binding-site residues to identify improvements in Clomeleon, a first-generation, suboptimal sensor. Structural analysis
revealed that these improvements involve halide contacts and distant side chain rearrangements. The development of optogenetic sensors
that respond to neural activity enables cellular tracking of neural activity using optical, rather than electrophysiological, signals. Construction of
such sensors using in vitro protein engineering establishes a powerful approach for developing new probes for brain imaging.
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