Directed Evolution of a Bright Near-Infrared Fluorescent Rhodopsin Using a Synthetic Chromophore.
By engineering a microbial rhodopsin, Archaerhodopsin-3 (Arch), to bind a synthetic chromophore, merocyanine retinal, in place of the natural chromophore all-trans-retinal (ATR), we generated a protein with exceptionally bright and unprecedentedly red-shifted near-infrared (NIR) fluorescence. We show that chromophore substitution generates a fluorescent Arch complex with a 200-nm bathochromic excitation shift relative to ATR-bound wild-type Arch and an emission maximum at 772 nm. Directed evolution of this complex produced variants with pH-sensitive NIR fluorescence and molecular brightness 8.5-fold greater than the brightest ATR-bound Arch variant. The resulting proteins are well suited to bacterial imaging; expression and stability have not been optimized for mammalian cell imaging. By targeting both the protein and its chromophore, we overcome inherent challenges associated with engineering bright NIR fluorescence into Archaerhodopsin. This work demonstrates an efficient strategy for engineering non-natural, tailored properties into microbial opsins, properties relevant for imaging and interrogating biological systems.
['Herwig|Lukas|L|', 'Rice|Austin J|AJ|', 'Bedbrook|Claire N|CN|', 'Zhang|Ruijie K|RK|', 'Lignell|Antti|A|', 'Cahn|Jackson K B|JKB|', 'Renata|Hans|H|', 'Dodani|Sheel C|SC|', 'Cho|Inha|I|', 'Cai|Long|L|', 'Gradinaru|Viviana|V|', 'Arnold|Frances H|FH|']
D001665:Binding Sites D019020:Directed Molecular Evolution D004926:Escherichia coli D006863:Hydrogen-Ion Concentration D007536:Isomerism D007700:Kinetics D008856:Microscopy, Fluorescence D062105:Molecular Docking Simulation D016297:Mutagenesis, Site-Directed D017434:Protein Structure, Tertiary D012172:Retinaldehyde D012243:Rhodopsin D019265:Spectroscopy, Near-Infrared
directed evolution near-infrared fluorescence Archaerhodopsin live-cell imaging protein engineering synthetic chromophore substitution