Articles pubs.acs.org/acschemicalbiology
Brighter Red Fluorescent Proteins by Rational Design of TripleDecker Motif Antonia T. Pandelieva,† Miranda J. Baran,† Guido F. Calderini,† Jenna L. McCann,† Véronique Tremblay,‡ Sabina Sarvan,‡ James A. Davey,† Jean-François Couture,*,‡,§ and Roberto A. Chica*,†,§ †
Department of Chemistry and Biomolecular Sciences, University of Ottawa, 10 Marie-Curie, Ottawa, Ontario K1N 6N5, Canada 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 ‡
S Supporting Information *
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. ed fluorescent proteins (RFPs) from Anthozoa have found widespread application in biological research. They are used as fusion tags to track proteins within cells,1 as partners in FRET pairs,2 and as signal transducers in biosensors.3−5 Because of their longer emission wavelengths (>570 nm), RFPs are particularly suited for whole-body imaging of research model animals, as red light is less toxic to cells and can penetrate deeper into tissues.6,7 For all applications of RFPs, high brightness is desirable. However, a drawback of monomeric RFPs is that they typically display lower brightness than green or yellow fluorescent proteins, and their brightness tends to decrease as their emission wavelength increases.6 Thus, there is still a need to engineer monomeric RFPs to maximize their brightness at longer emission wavelengths. RFP brightness is defined as the product of their molar extinction coefficient and quantum yield.8 The extinction coefficient relates to the efficiency with which the chromophore can absorb light. However, reported values of molar extinction coefficients are also dependent on chromophore maturation efficiency since mature RFP samples can contain a mixture of green and red chromophores,9,10 and protein quantification methods cannot distinguish molecules containing red chromo-
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phores from those that are misfolded or that contain a green chromophore in the RFP population in solution.11 Extinction coefficients of most monomeric RFPs are in the 40−100 mM−1 cm−1 range,6,12,13 which is similar to that of Aequorea victoria green fluorescent protein (GFP) and its variants. On the other hand, quantum yield relates to the efficiency with which the chromophore emits fluorescence and is defined as the number of photons emitted over the number of photons absorbed. Although monomeric RFPs can have quantum yields up to 0.49,14 most display quantum yields lower than 0.30, making them significantly less bright than monomeric green and yellow fluorescent proteins, which predominantly have quantum yields in the 0.60−0.80 range.6,12,13 To increase the brightness of monomeric RFPs, researchers have traditionally used directed evolution methods whereby large libraries of random mutants (103−106) are screened using high-throughput fluorescence-based assays. Although brighter Received: September 24, 2015 Accepted: December 23, 2015 Published: December 23, 2015 508
DOI: 10.1021/acschembio.5b00774 ACS Chem. Biol. 2016, 11, 508−517
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ACS Chemical Biology
of amino-acid substitutions that cause emission wavelength bathochromic shifts without destabilizing the protein fold.20 mRojoA displays a 22 nm bathochromic shift relative to its parent mCherry and a quantum yield of 0.02, 10 times lower than that of the parent. This lower quantum yield is mostly caused by the I197Y point mutation that reduces quantum yield to 0.03 and also red-shifts the emission wavelength by 7 nm when introduced alone into mCherry.20 mRojoA was selected as the test protein for our hypothesis because it already contains an aromatic residue (Tyr197) that is π-stacked with the chromophore and because its low quantum yield can be further optimized. Structure-Based Rational Design. Inspection of the crystal structure of mRojoA (PDB ID: 3NEZ20) shows that a second aromatic residue could be introduced at position 63, which is located directly above the chromophore (Figure 1). In
monomeric RFPs have been engineered using these approaches, multiple rounds of mutagenesis followed by screening were required to achieve 2- to 3-fold increases in brightness.15−17 Unsurprisingly, much of the observed brightness increases are due to improvements in chromophore maturation efficiency that result in higher molar extinction coefficients,15,16 but only modest quantum yield increases (
