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Directed Evolution of a Bright Near-Infrared Fluorescent Rhodopsin Using a Synthetic Chromophore

Herwig, Lukas and Rice, Austin J. and Bedbrook, Claire N. and Zhang, Ruijie K. and Lignell, Antti and Cahn, Jackson K. B. and Renata, Hans and Dodani, Sheel C. and Cho, Inha and Cai, Long and Gradinaru, Viviana and Arnold, Frances H. (2017) Directed Evolution of a Bright Near-Infrared Fluorescent Rhodopsin Using a Synthetic Chromophore. Cell Chemical Biology, 24 (3). pp. 415-425. ISSN 2451-9456. PMCID PMC5357175. doi:10.1016/j.chembiol.2017.02.008.

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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.

Item Type:Article
Related URLs:
URLURL TypeDescription CentralArticle
Bedbrook, Claire N.0000-0003-3973-598X
Zhang, Ruijie K.0000-0002-7251-5527
Lignell, Antti0000-0001-7664-5583
Renata, Hans0000-0003-2468-2328
Cho, Inha0000-0002-7564-5378
Cai, Long0000-0002-7154-5361
Gradinaru, Viviana0000-0001-5868-348X
Arnold, Frances H.0000-0002-4027-364X
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 ( 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.
Group:Resnick Sustainability Institute, Caltech Center for Environmental Microbial Interactions (CEMI), Rosen Bioengineering Center
Funding AgencyGrant Number
Swiss National Science Foundation (SNSF)P2BSP3_151863
NIH Predoctoral FellowshipF32GM116319
NIH Predoctoral FellowshipF31MH102913
NSF Graduate Research FellowshipDGE-1144469
Donna and Benjamin M. Rosen Bioengineering CenterUNSPECIFIED
Resnick Sustainability InstituteUNSPECIFIED
Army Research Office (ARO)W911F-09-0001
National Institute of Mental Health (NIMH)UNSPECIFIED
Subject Keywords:synthetic chromophore substitution; directed evolution; protein engineering; near-infrared fluorescence; Archaerhodopsin; live-cell imaging
Issue or Number:3
PubMed Central ID:PMC5357175
Record Number:CaltechAUTHORS:20170307-082231020
Persistent URL:
Official Citation:Lukas Herwig, Austin J. Rice, Claire N. Bedbrook, Ruijie K. Zhang, Antti Lignell, Jackson K.B. Cahn, Hans Renata, Sheel C. Dodani, Inha Cho, Long Cai, Viviana Gradinaru, Frances H. Arnold, Directed Evolution of a Bright Near-Infrared Fluorescent Rhodopsin Using a Synthetic Chromophore, Cell Chemical Biology, Volume 24, Issue 3, 16 March 2017, Pages 415-425, ISSN 2451-9456,
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:74829
Deposited By: Tony Diaz
Deposited On:07 Mar 2017 18:05
Last Modified:11 Nov 2021 05:30

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