... we compared the relative signaling efficiency of different PAFPs by determining the number of single-molecule localizations per cell of the PAFPs fused to a common target protein under endogenous expression. We fused the PAFPs to the E. coli gene hupA, which encodes a subunit of the nucleoid-associated protein HU, at its endogenous chromosomal locus and determined the total number of HU-PAFP localizations per cell. Among the red PAFPs, mMaple provided by far the largest number of HU localizations, which was 6- to 32-fold higher than those of other HU-PAFP fusion proteins.
... our results show that mMaple has a much higher signaling efficiency than Dendra2, mEos2, mEos3.2, tdEos, mKikGR, PAmCherry, and PATagRFP. However, the dimerization tendency of mMaple could lead to aggregation effects on the target proteins. Dendra2, mEos3.2, and PATagRFP exhibit undetectable dimerization tendency, but have low signaling efficiency. It is thus desirable to develop a new PAFP that has both high signaling efficiency and low dimerization tendency. To this end, we engineered two new PAFPs by introducing point mutations into mMaple designed to destabilize the dimerization of this protein.
We first took inspiration from the two mutations that make mEos3.2 more monomeric than mEos2: I102N and Y189A (15). Based on a sequence alignment between mEos2 and mMaple, we made the comparable mutations, I111N and Y198A, in mMaple... mMaple2 exhibited a similar photon budget, on–off ratio, and signaling efficiency as those of mMaple. ClpP-mMaple2 proteins still formed puncta in some cells. However, the percentage of cells showing single punctum was significantly reduced in comparison with ClpP-mMaple, suggesting a lower dimerization tendency of mMaple2.
To further reduce the residual dimerization tendency of mMaple2, we screened a series of mutations to residues that are predicted to be solvent-exposed near residues 111 and 198. We focused on charged residues and switched them to the opposite charge. One of the derivatives, with mutations E82R, D83K, and D197K, in addition to I111N and Y198A, exhibited undetectable dimerization tendency when fused to ClpP. This derivative, which we termed mMaple3, again had a similar photon budget to that of mMaple and even a lower on–off ratio compared with mMaple. Its signaling efficiency was only moderately reduced from those of mMaple or mMaple2.