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Published November 27, 2018 | Supplemental Material + Published + Submitted
Journal Article Open

Monomerization of Far-Red Fluorescent Proteins


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.

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, www.wwpdb.org (PDB ID code: 6DEJ). GenBank IDs [accession nos. MK040729 (mGinger), MK040730 (mGinger2), MK040731 (mKelly1), and MK040732 (mKelly2)]. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1807449115/-/DCSupplemental.

Attached Files

Published - E11294.full.pdf

Submitted - 162842.full.pdf

Supplemental Material - pnas.1807449115.sapp.pdf


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August 22, 2023
October 18, 2023