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TitleMonomerization of far-red fluorescent proteins.
Publication TypeJournal Article
Year of Publication2018
AuthorsWannier, Timothy M., Sarah K. Gillespie, Nicholas Hutchins, Scott R McIsaac, Sheng-Yi Wu, Yi Shen, Robert E. Campbell, Kevin S. Brown, and Stephen L. Mayo
JournalProc Natl Acad Sci U S A
Volume115
Issue48
PaginationE11294-E11301
Date Published2018 11 27
ISSN1091-6490
Abstract

-class red fluorescent proteins (RFPs) are frequently used as biological markers, with far-red (λ ∼ 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 = 656 nm), which, along with the recently reported mGarnet2 [Matela G, et al. (2017) 53:979-982], forms a class of bright, monomeric, far-red FPs.

DOI10.1073/pnas.1807449115
Alternate JournalProc. Natl. Acad. Sci. U.S.A.
PubMed ID30425172
PubMed Central IDPMC6275547
Grant ListR21 EB018579 / EB / NIBIB NIH HHS / United States