Directed Evolution of a Bright Near-Infrared Fluorescent Rhodopsin Using a Synthetic Chromophore
Abstract
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.
Additional Information
© 2017 Elsevier Ltd. Received 1 July 2016, Revised 28 November 2016, Accepted 1 February 2017, Available online 2 March 2017. Published: March 2, 2017. The authors would like to thank Sabine Brinkmann-Chen and Andrew Buller for critical review of the manuscript and David VanderVelde for assistance with NMR analysis. L.H. was supported by a fellowship from the Swiss National Science Foundation (SNSF; P2BSP3_151863). The Ruth L. Kirschstein National Research Service Award supports A.J.R. (F32GM116319), C.N.B. (F31MH102913), and S.C.D. (5F32GM106618). R.K.Z. was supported by a National Science Foundation Graduate Research Fellowship (NSF GRFP; DGE-1144469), is a trainee in the Caltech Biotechnology Leadership Program, and has received financial support from the Donna and Benjamin M. Rosen Bioengineering Center. J.K.B.C. acknowledges the support of the Resnick Sustainability Institute (Caltech). Research is supported by the National Center for Research Resources, ARRA SIG Program S10RR027203 (F.H.A.); National Institute of Mental Health R21MH103824 (V.G. and F.H.A.); and the Institute for Collaborative Biotechnologies through grant number W911F-09-0001 from the US Army Research Office (F.H.A.). The authors would like to acknowledge the Beckman Institute for the Resource Center on CLARITY, Optogenetics, and Vector Engineering for technology development and broad dissemination (http://www.beckmaninstitute.caltech.edu/clover.shtml). The content is solely the responsibility of the authors and does not necessarily reflect the position or policy of the National Center for Research Resources, the NIH, or the Government, and no official endorsement should be inferred.Attached Files
Accepted Version - nihms850709.pdf
Supplemental Material - mmc1.pdf
Supplemental Material - mmc2.zip
Files
Additional details
- PMCID
- PMC5357175
- Eprint ID
- 74829
- DOI
- 10.1016/j.chembiol.2017.02.008
- Resolver ID
- CaltechAUTHORS:20170307-082231020
- P2BSP3_151863
- Swiss National Science Foundation (SNSF)
- F32GM116319
- NIH Predoctoral Fellowship
- F31MH102913
- NIH Predoctoral Fellowship
- 5F32GM106618
- NIH
- DGE-1144469
- NSF Graduate Research Fellowship
- Donna and Benjamin M. Rosen Bioengineering Center
- Resnick Sustainability Institute
- S10RR027203
- NIH
- R21MH103824
- NIH
- W911F-09-0001
- Army Research Office (ARO)
- National Institute of Mental Health (NIMH)
- Created
-
2017-03-07Created from EPrint's datestamp field
- Updated
-
2021-11-11Created from EPrint's last_modified field
- Caltech groups
- Resnick Sustainability Institute, Caltech Center for Environmental Microbial Interactions (CEMI), Rosen Bioengineering Center