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5H89

Crystal structure of mRojoA mutant - T16V - P63Y - W143G - L163V

Summary for 5H89
Entry DOI10.2210/pdb5h89/pdb
Related5H87 5H88
DescriptormRojoA fluorescent protein (2 entities in total)
Functional Keywordsquantum yield, protein engineering, fluorescent protein
Biological sourceDiscosoma sp.
Total number of polymer chains4
Total formula weight110504.20
Authors
Pandelieva, A.T.,Tremblay, V.,Sarvan, S.,Chica, R.A.,Couture, J.-F. (deposition date: 2015-12-23, release date: 2016-01-27, Last modification date: 2024-10-30)
Primary citationPandelieva, A.T.,Baran, M.J.,Calderini, G.F.,McCann, J.L.,Tremblay, V.,Sarvan, S.,Davey, J.A.,Couture, J.F.,Chica, R.A.
Brighter Red Fluorescent Proteins by Rational Design of Triple-Decker Motif.
Acs Chem.Biol., 11:508-517, 2016
Cited by
PubMed Abstract: Red fluorescent proteins (RFPs) are used extensively in chemical biology research as fluorophores for live cell imaging, as partners in FRET pairs, and as signal transducers in biosensors. For all of these applications, brighter RFP variants are desired. Here, we used rational design to increase the quantum yield of monomeric RFPs in order to improve their brightness. We postulated that we could increase quantum yield by restricting the conformational degrees of freedom of the RFP chromophore. To test our hypothesis, we introduced aromatic residues above the chromophore of mRojoA, a dim RFP containing a π-stacked Tyr residue directly beneath the chromophore, in order to reduce chromophore conformational flexibility via improved packing and steric complementarity. The best mutant identified displayed an absolute quantum yield increase of 0.07, representing an over 3-fold improvement relative to mRojoA. Remarkably, this variant was isolated following the screening of only 48 mutants, a library size that is several orders of magnitude smaller than those previously used to achieve equivalent gains in quantum yield in other RFPs. The crystal structure of the highest quantum yield mutant showed that the chromophore is sandwiched between two Tyr residues in a triple-decker motif of aromatic rings. Presence of this motif increases chromophore rigidity, as evidenced by the significantly reduced temperature factors compared to dim RFPs. Overall, the approach presented here paves the way for the rapid development of fluorescent proteins with higher quantum yield and overall brightness.
PubMed: 26697759
DOI: 10.1021/acschembio.5b00774
PDB entries with the same primary citation
Experimental method
X-RAY DIFFRACTION (1.76 Å)
Structure validation

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