Grimley, Joshua S. Li, Li Wang, Weina Wen, Lei Beese, Lorena S. Hellinga, Homme W. Augustine, George J. 2024-01-20T11:30:22Z 2024-01-20T11:30:22Z 2022-01-10 2013-10-09 0270-6474 https://pubs.kist.re.kr/handle/201004/127548 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. English SOC NEUROSCIENCE GREEN FLUORESCENT PROTEIN CHLORIDE INDICATOR BRAIN MECHANISM VARIANTS BINDING SENSITIVITY TRANSIENTS CYAN Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation Article 10.1523/JNEUROSCI.4616-11.2013 1 JOURNAL OF NEUROSCIENCE, v.33, no.41, pp.16297 - 16309 JOURNAL OF NEUROSCIENCE 33 41 16297 16309 scie scopus 000325644800024 2-s2.0-84885095868 Neurosciences Neurosciences & Neurology Article GREEN FLUORESCENT PROTEIN CHLORIDE INDICATOR BRAIN MECHANISM VARIANTS BINDING SENSITIVITY TRANSIENTS CYAN