Figure 1. Development and characterization of three monomeric miRFPs.

(a) Schematics of directed molecular evolution resulted in three monomeric miRFPs. The chromophore-binding PAS-GAF domains, which are not involved in dimerization of RpBphP1, were used as a starting point. To exclude formation of even weak dimers, we mutated residues in the C-terminal α-helix in the GAF domain. To obtain spectrally distinct variants, we mutated residues 201 and 202 in the -PXSDIP- motif and residue 253 in the -PIH- motif in the GAF domain. (b) Fluorescence excitation spectra of engineered miRFP670, miRFP703 and miRFP709. (c) Fluorescence emission spectra of miRFPs. (d) Size exclusion chromatography of miRFPs and indicated molecular weight standards. Apparent molecular weight of all miRFPs was ∼35 kDa. (e) Brightness of live HeLa cells transiently transfected with several BphP-based NIR FPs analysed by flow cytometry. The NIR fluorescence intensity was normalized to transfection efficiency (fluorescence of co-transfected EGFP), to excitation efficiency of each FP with 635 nm laser, and to fluorescence signal of each FP in the emission filter. The NIR effective brightness of miRFP670 was assumed to be 100%. Error bars, s.d. (n=3; transfection experiments). (f) Representative fluorescence images of several BphP-based NIR FPs in live HeLa cells. Acquisition time for each image is indicated. Scale bar, 10 μm.