Figure 3. Cherry is the optimal red FRET acceptor for yellow donors.
a. The sequences of YPet-red constructs were aligned using Clustal Omega, shown here around the linker region, with beta barrel residues of the FPs highlighted. The remaining residues have a predicted coiled secondary structure. Notice the additional residues (8-11 DNMA) in mCherry compared to the other red proteins. In order to make a robust comparison of FRET efficiencies, constructs denoted as YPet-RedL or Cit-RedL have DNMA inserted in Ruby2, TagRFP and TagRFPt. In YPet-RedS or Cit-RedS DNMA has been deleted in mCherry. b. In COS-7 cells expressing YPet-CherryL (WT), the acceptor was selectively photobleached by collimated 561 nm laser illumination (4,500 ms pulse at 95 W/cm2; 15 cycles). c. The average intensity for ROIs during the acquisition resulting in a FRET efficiency of 0.35 ± 0.06. d. Characteristic FRET efficiencies for various yellow/red pairs, all error bars are standard deviation with N ≥ 10. YPet-Red bars not connected by the same letter are significantly different (Note: No comparison is made across donor at this point) (p < 0.05) using One-Way ANOVA followed by Tukey HSD post hoc comparison of means. Asterisk indicates significant difference between YPet and corresponding Citrine construct (p < 0.05) using Two-Way ANOVA followed by Tukey HSD post hoc comparison of means. e. Characteristic FRET efficiencies measured by acceptor photobleaching for YPet-Red constructs following treatment with 100 μg/mL CHX for 5 hrs. f. N-Way FRET analysis of various YPet-Red constructs following CHX treatment demonstrating the differential maturation rates of the FPs. mCherry-YPet has a higher control ratio of 0.8 compared to mRuby2-YPet, which is 0.4. Following treatment with CHX for 5 hours, the [R] to [Y] ratio is 1 to 1 for all constructs except YPet-Ruby2L which increases to 0.8 (p < 0.05) Two-way ANOVA followed by Tukey HSD post hoc comparison of means. (Significance only shown for molar ratios of wildtype constructs for clarity.)