7RY2

mSandy2

Summary for 7RY2

• Entry DOI: 10.2210/pdb7ry2/pdb
• Descriptor: mSandy2 (3 entities in total)
• Functional Keywords: red fluorescent protein, discosoma sp., monomeric, fluorescent protein
• Biological source: Discosoma sp.; More
• Total number of polymer chains: 8
• Total formula weight: 221013.44

Authors

Chica, R.A.,Legault, S.,Thompson, M.C. (deposition date: 2021-08-24, release date: 2022-01-26, Last modification date: 2023-11-15)

Primary citation

Legault, S.,Fraser-Halberg, D.P.,McAnelly, R.L.,Eason, M.G.,Thompson, M.C.,Chica, R.A.
Generation of bright monomeric red fluorescent proteins via computational design of enhanced chromophore packing.
Chem Sci, 13:1408-1418, 2022

PubMed 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 ( = 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 ( = 606 nm, quantum yield = 0.35), the brightest sp. derived monomeric RFP with an emission maximum above 600 nm reported to date. Crystallographic analysis of mSandy2 showed that the chromophore -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.

PubMed: 35222925
DOI: 10.1039/d1sc05088e

Experimental method

X-RAY DIFFRACTION (2.05 Å)