Table 1.
FRET efficiencies obtained with mTq2 variants measured by fluorometry.
| Donor FP to mNG | Fusion position | EfA (%) | SD | Repeats | |
|---|---|---|---|---|---|
| Cytoplasmic FRET | |||||
| Positive control tandems | |||||
| mNG‐mTq2 | mTq2 | Free floating | 61.3 | 6.4 | 4 |
| mNG‐sfTq2 | sfTq2 | Free floating | 64.5 | 3.0 | 7 |
| mNG‐sfTq2C70V | sfTq2ox | Free floating | 66.4 | 3.1 | 8 |
| Periplasmic FRET | |||||
| Positive control tandems | |||||
| mNG‐sfTq2 | sfTq2 | OmpA177 (OM) | 18.8 | 2.1 | 6 |
| mNG‐sfTq2C48S | C48S | OmpA177 | 32.9 | 1.9 | 2 |
| mNG‐sfTq2C70V | sfTq2ox | OmpA177 | 39.1 | 3.8 | 11 |
| mNG‐sfTq2C48S‐C70V | C48S‐C70V | OmpA177 | 40.3 | 1.2 | 2 |
| mNG‐sfTq2C70V | sfTq2ox | OmpA177* | 42.5 | 3.1 | 6 |
| mNG‐sfTq2C70V | sfTq2ox | LpoB73 (OM) | 39.8 | 2.1 | 5 |
| mNG‐sfTq2C70V | sfTq2ox | NlpAss (IM) | 41.2 | 2.4 | 7 |
| mNG‐sfTq2C70V | sfTq2ox | MalFss‐mss (IM) | 42.9 | 2.5 | 3 |
| Negative controls | |||||
| mNG‐sfTq2 (IM‐OM) | sfTq2 | PBP5, OmpA177 | –1.4 | 1.4 | 4 |
| mNG‐sfTq2C70V (IM‐OM) | sfTq2ox | PBP5, OmpA177 | –1.3 | 2.2 | 13 |
| mNG‐sfTq2C70V (IM, IM) | sfTq2ox | NlpAss, FtsB | –0.7 | 1.7 | 10 |
| Biological interactions | |||||
| PBP5 + PBP5 | sfTq2 | IM, IM | 4.2 | 2.7 | 5 |
| PBP5S44G + PBP5S44G | sfTq2 | IM, IM | 8.8 | 4.2 | 6 |
| FtsB + FtsL | sfTq2ox | IM, IM | 19.2 | 4.5 | 8 |
| FtsL + FtsB | sfTq2ox | IM, IM | 15.6 | 2.6 | 8 |
| FtsLm4 + FtsB | sfTq2ox | IM, IM | 3.1 | 2.1 | 3 |
| FtsLm4 + FtsBm4 | sfTq2ox | IM, IM | 3.0 | 3.4 | 3 |
SS, signal sequence; mss, membrane spanning sequence, LpoB73 indicates residue 1‐73 of LpoB, OmpA177 indicates residue 1‐177 of OmpA, * indicates a different linker between mNG and sfTq2ox (EF instead of EL due to differences in cloning). Table S2 shows the FRET efficiencies for the new mTq2 variants measured with the plate reader. Representative unmixing data are shown in Fig. S14; the plate reader unmixing data are shown in Fig. S15.