Journal
CELL CHEMICAL BIOLOGY
Volume 24, Issue 3, Pages 415-425Publisher
CELL PRESS
DOI: 10.1016/j.chembiol.2017.02.008
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Funding
- Swiss National Science Foundation (SNSF) [P2BSP3_151863]
- Ruth L. Kirschstein National Research Service Award [F32GM116319, F31MH102913, 5F32GM106618]
- National Science Foundation Graduate Research Fellowship (NSF GRFP) [DGE-1144469]
- Donna and Benjamin M. Rosen Bioengineering Center
- Resnick Sustainability Institute (Caltech)
- National Center for Research Resources
- ARRA SIG Program [S10RR027203]
- National Institute of Mental Health [R21MH103824]
- Institute for Collaborative Biotechnologies through US Army Research Office [911F-09-0001]
- Swiss National Science Foundation (SNF) [P2BSP3_151863] Funding Source: Swiss National Science Foundation (SNF)
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By engineering a microbial rhodopsin, Archaerho-dopsin-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.
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