title: Brighter Red Fluorescent Proteins by Rational Design of Triple-Decker Motif.
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.
authors.affiliation: Department of Chemistry and Biomolecular Sciences, University of Ottawa , 10 Marie-Curie, Ottawa, Ontario K1N 6N5, Canada.
authors.forename: Antonia T
authors.initials: AT
authors.lastname: Pandelieva
authors.affiliation: Department of Chemistry and Biomolecular Sciences, University of Ottawa , 10 Marie-Curie, Ottawa, Ontario K1N 6N5, Canada.
authors.forename: Miranda J
authors.initials: MJ
authors.lastname: Baran
authors.affiliation: Department of Chemistry and Biomolecular Sciences, University of Ottawa , 10 Marie-Curie, Ottawa, Ontario K1N 6N5, Canada.
authors.forename: Guido F
authors.initials: GF
authors.lastname: Calderini
authors.affiliation: Department of Chemistry and Biomolecular Sciences, University of Ottawa , 10 Marie-Curie, Ottawa, Ontario K1N 6N5, Canada.
authors.forename: Jenna L
authors.initials: JL
authors.lastname: McCann
authors.affiliation: Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology, University of Ottawa , 451 Smyth Road, Ottawa, Ontario K1H 8M5, Canada.
authors.forename: Véronique
authors.initials: V
authors.lastname: Tremblay
authors.affiliation: Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology, University of Ottawa , 451 Smyth Road, Ottawa, Ontario K1H 8M5, Canada.
authors.forename: Sabina
authors.initials: S
authors.lastname: Sarvan
authors.affiliation: Department of Chemistry and Biomolecular Sciences, University of Ottawa , 10 Marie-Curie, Ottawa, Ontario K1N 6N5, Canada.
authors.forename: James A
authors.initials: JA
authors.lastname: Davey
authors.affiliation: Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology, University of Ottawa , 451 Smyth Road, Ottawa, Ontario K1H 8M5, Canada.|Centre for Catalysis Research and Innovation, University of Ottawa , 30 Marie-Curie, Ottawa, Ontario K1N 6N5, Canada.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Couture
authors.affiliation: Department of Chemistry and Biomolecular Sciences, University of Ottawa , 10 Marie-Curie, Ottawa, Ontario K1N 6N5, Canada.|Centre for Catalysis Research and Innovation, University of Ottawa , 30 Marie-Curie, Ottawa, Ontario K1N 6N5, Canada.
authors.forename: Roberto A
authors.initials: RA
authors.lastname: Chica
mesh_terms: D000818:Animals; D037421:Anthozoa / Q000737:chemistry* / Q000235:genetics; D018360:Crystallography, X-Ray; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D000097573:Red Fluorescent Protein
chemical_list: D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein