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

Abstract

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 (λem = 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 (λem = 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.

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

Supplementary files

Article information

Article type
Edge Article
Submitted
14 Sep 2021
Accepted
07 Jan 2022
First published
11 Jan 2022
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY-NC license

Chem. Sci., 2022, Advance Article

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

S. Legault, D. P. Fraser-Halberg, R. L. McAnelly, M. G. Eason, M. C. Thompson and R. A. Chica, Chem. Sci., 2022, Advance Article , DOI: 10.1039/D1SC05088E

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