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.

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Dans les débats entourant l'augmentation biomédicale de la moralité humaine, il est largement affirmé que la moralité a été façonnée au cours de l'évolution pour être rigidement tribale. Allen Buchanan remet en question cette hypothèse en faisant valoir qu'une moralité plastique qui répond à l'environnement serait évolutionnellement privilégiée, et donc que la meilleure façon de modifier la moralité humaine à l'avenir ne serait pas par des interventions biomédicales, mais par la conception de meilleures institutions.In debates surrounding the biomedical enhancement of human morality, it is widely believed that morality was evolutionarily shaped to be rigidly tribal. Allen Buchanan challenges this assumption by making the case that a plastic morality that responds to our environment would be evolutionarily favored, and thus the best way to shape human morality going forward would not be through biomedical interventions, but by designing better institutions.