Generation of bright monomeric red fluorescent proteins via computational design of enhanced chromophore packing

Legault, Sandrine; Fraser-Halberg, Derek P.; McAnelly, Ralph L.; Eason, Matthew G.; Thompson, Michael C.; Chica, Roberto A.

doi:10.1039/d1sc05088e

Title: Generation of bright monomeric red fluorescent proteins via computational design of enhanced chromophore packing

Journal Article · 10 January 2022 · Chemical Science

DOI: https://doi.org/10.1039/d1sc05088e · OSTI ID:1903860

Legault, Sandrine ^[1];

^[2]; McAnelly, Ralph L. ^[3]; Eason, Matthew G. ^[2]; Thompson, Michael C. ^[3];

^[2]

University of Ottawa, ON (Canada); OSTI
University of Ottawa, ON (Canada)
University of California, Merced, CA (United States)

Red fluorescent proteins (RFPs) have found widespread application in chemical and biological research due to their longer emission wavelengths. Here, we use computational protein design to increase the quantum yield and thereby brightness of a dim monomeric RFP (mRojoA, quantum yield = 0.02) by optimizing chromophore packing with aliphatic residues, which we hypothesized would reduce torsional motions causing non-radiative decay. Experimental characterization of the top 10 designed sequences yielded mSandy1 (λ_em = 609 nm, quantum yield = 0.26), a variant with equivalent brightness to mCherry, a widely used RFP. We next used directed evolution to further increase brightness, resulting in mSandy2 (λ_em = 606 nm, quantum yield = 0.35), the brightest Discosoma sp. derived monomeric RFP with an emission maximum above 600 nm reported to date. Crystallographic analysis of mSandy2 showed that the chromophore p-hydroxybenzylidene moiety is sandwiched between the side chains of Leu63 and Ile197, a structural motif that has not previously been observed in RFPs, and confirms that aliphatic packing leads to chromophore rigidification. Our results demonstrate that computational protein design can be used to generate bright monomeric RFPs, which can serve as templates for the evolution of novel far-red fluorescent proteins.

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Research Organization:: University of California, Merced, CA (United States); University of Ottawa, ON (Canada)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER); Canada Foundation for Innovation; Natural Sciences and Engineering Research Council of Canada; Human Frontier Science Program; National Institutes of Health (NIH)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1903860

Journal Information:: Chemical Science, Journal Name: Chemical Science Journal Issue: 5 Vol. 13; ISSN 2041-6520

Publisher:: Royal Society of ChemistryCopyright Statement

Country of Publication:: United States

Language:: English

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