Fig. 1: The rational mutagenesis model and the resulting enhanced spectroscopic characteristics of stagRFP compared to TagRFP-T.
From: A rationally enhanced red fluorescent protein expands the utility of FRET biosensors
a Schematic underlying the rational engineering approach. A model system bearing high FLINC state reveals the presence of the external acidic residue that weakens the TagRFP-T chromophore. Site-directed mutagenesis products that rescue the model back to a low FLINC state harbor mutants with improved insulation and stable fluorescence. Finally, removal of Dronpa confirms the improved behavior of the mutant TagRFP-T. A ribbon structure and APBS electrostatic surface of TagRFP-T (based on PDB 3T6H) demonstrates the large acidic (red) solvent exposed surface opposite the chromophore. The location of D159 is indicated by dashed circles. Normalized in vitro absorption (b) and fluorescence (c) spectra of purified stagRFP compared to TagRFP-T demonstrating the significantly higher extinction coefficient and quantum yield. d Cyclic excitation of HeLa cells expressing different TagRFP variants show photoactivation is present in parent proteins (TagRFP and TagRFP-T), but not the new mutant stagRFP (TagRFP, n = 9 biologically independent cells; TagRFP-T, n = 10; and stagRFP, n = 12 biologically independent cells); each cell is represented by the, respectively, colored symbol, and the average values across all cells are shown using solid lines. Source data for b–c are provided as a Source Data file.
