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Monomerization of Far-Red Fluorescent Proteins

Wannier, Timothy M. and Gillespie, Sarah K. and Hutchins, Nicholas and McIsaac, R. Scott and Wu, Sheng-Yi and Shen, Yi and Campbell, Robert E. and Brown, Kevin S. and Mayo, Stephen L. (2018) Monomerization of Far-Red Fluorescent Proteins. Proceedings of the National Academy of Sciences of the United States of America, 115 (48). E11294-E11301. ISSN 0027-8424. PMCID PMC6275547.

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Anthozoa-class red fluorescent proteins (RFPs) are frequently used as biological markers, with far-red (λ_(em) ∼ 600–700 nm) emitting variants sought for whole-animal imaging because biological tissues are more permeable to light in this range. A barrier to the use of naturally occurring RFP variants as molecular markers is that all are tetrameric, which is not ideal for cell biological applications. Efforts to engineer monomeric RFPs have typically produced dimmer and blue-shifted variants because the chromophore is sensitive to small structural perturbations. In fact, despite much effort, only four native RFPs have been successfully monomerized, leaving the majority of RFP biodiversity untapped in biomarker development. Here we report the generation of monomeric variants of HcRed and mCardinal, both far-red dimers, and describe a comprehensive methodology for the monomerization of red-shifted oligomeric RFPs. Among the resultant variants is mKelly1 (emission maximum, λ_(em) = 656 nm), which, along with the recently reported mGarnet2 [Matela G, et al. (2017) Chem Commun (Camb) 53:979–982], forms a class of bright, monomeric, far-red FPs.

Item Type:Article
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URLURL TypeDescription Information Paper CentralArticle
McIsaac, R. Scott0000-0002-5339-6032
Mayo, Stephen L.0000-0002-9785-5018
Additional Information:© 2018 National Academy of Sciences. Published under the PNAS license. Contributed by Stephen L. Mayo, October 12, 2018 (sent for review June 13, 2018; reviewed by Amy E. Palmer and Vladislav V. Verkhusha). PNAS published ahead of print November 13, 2018. We thank Yun Mou, Matthew Moore, and Roberto Chica for many helpful conversations. We would also like to thank Prof. Frances Arnold for her advice and support. Jens Kaiser and Pavle Nikolovski were generous with their time and advice in troubleshooting protein crystallography. The authors are grateful for the use of the beamline 12-2 at the Stanford Synchrotron Radiation Lightsource and to the Gordon and Betty Moore Foundation for support of the Molecular Observatory at the California Institute of Technology. We also acknowledge the funding and support of the National Institute of Biomedical Imaging and Bioengineering (Grant R21EB018579). Author contributions: T.M.W., R.S.M., R.E.C., K.S.B., and S.L.M. designed research; T.M.W., S.K.G., N.H., S.-Y.W., and Y.S. performed research; T.M.W., Y.S., and K.S.B. analyzed data; and T.M.W. and S.L.M. wrote the paper. Reviewers: A.E.P., University of Colorado; and V.V.V., Albert Einstein College of Medicine. The authors declare no conflict of interest. Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, (PDB ID code: 6DEJ). GenBank IDs [accession nos. MK040729 (mGinger), MK040730 (mGinger2), MK040731 (mKelly1), and MK040732 (mKelly2)]. This article contains supporting information online at
Funding AgencyGrant Number
Gordon and Betty Moore FoundationUNSPECIFIED
Subject Keywords:fluorescent protein; red fluorescent protein; protein engineering; computational protein design; RFP
Issue or Number:48
PubMed Central ID:PMC6275547
Record Number:CaltechAUTHORS:20180105-112949227
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Official Citation:Monomerization of far-red fluorescent proteins. Timothy M. Wannier, Sarah K. Gillespie, Nicholas Hutchins, R. Scott McIsaac, Sheng-Yi Wu, Yi Shen, Robert E. Campbell, Kevin S. Brown, Stephen L. Mayo. Proceedings of the National Academy of Sciences Nov 2018, 115 (48) E11294-E11301; DOI: 10.1073/pnas.1807449115
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:84127
Deposited By: George Porter
Deposited On:05 Jan 2018 20:45
Last Modified:07 Apr 2020 23:04

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