TABLE 1.
Melting temperatures of AbTetR variants in the absence or presence of various tetracycline antibiotics.
| Mutant | Apo | Chl | ΔTm | ΔΔTm | Dem | ΔTm | ΔΔTm | Dox | ΔTm | ΔΔTm | Mec | ΔTm | ΔΔTm | Met | ΔTm | ΔΔTm | Min | ΔTm | ΔΔTm | Oxy | ΔTm | ΔΔTm | Tet | ΔTm | ΔΔTm | Tig* | ΔTm | ΔΔTm |
| Wildtype | 45.6 ± 0.2 | 62.3± 0.3 | 16.7 | − | 62.6 ± 0.1 | 17.0 | − | 66.4± 0.2 | 20.8 | − | 67.7± 0.3 | 22.1 | − | 66.0± 0.3 | 20.4 | − | 57.1± 0.3 | 11.5 | − | 65.5± 0.3 | 19.9 | − | 63.0± 0.3 | 17.4 | − | 56.6± 0.3 | 11.0 | − |
| H64A | n.d. | 45.5± 0.4 | n.d. | n.d. | 46.4± 0.3 | n.d. | n.d. | 47.7± 0.2 | n.d. | n.d. | 51.1± 0.5 | n.d. | n.d. | 47.4± 0.2 | n.d. | n.d. | n.d. | n.d. | n.d. | 45.6± 0.2 | n.d. | n.d. | 44.8 ± 0.5 | n.d. | n.d. | 39.7± 0.9 | n.d. | n.d. |
| N82A | 41.5± 0.3 | 43.5± 0.3 | 2.0 | −14.7 | 45.7± 0.3 | 4.2 | −12.8 | 42.8± 0.2 | 1.3 | −19.5 | 47.1± 0.2 | 5.6 | −16.5 | 44.1± 0.2 | 2.6 | −17.8 | 42.4± 0.2 | 0.9 | −10.6 | 42.4± 0.2 | 0.9 | −19.0 | 42.3± 0.2 | 0.8 | −16.6 | n.d. | n.d. | n.d. |
| F86A | n.d. | 45.1± 0.3 | n.d. | n.d. | 45.9± 0.1 | n.d. | n.d. | 49.1± 0.3 | n.d. | n.d. | 54.2± 0.1 | n.d. | n.d. | 48.3± 0.2 | n.d. | n.d. | n.d. | n.d. | n.d. | 46.6± 0.1 | n.d. | n.d. | 45.6± 0.4 | n.d. | n.d. | 36.9± 0.7 | n.d. | n.d. |
| H100A | 45.9± 0.2 | 57.3± 0.2 | 11.4 | −5.3 | 58.7± 0.1 | 12.8 | −4.2 | 60.9± 0.2 | 15.0 | −5.8 | 64.8± 0.1 | 18.9 | −3.2 | 60.8± 0.1 | 14.9 | −5.5 | 49.1± 0.5 | 3.2 | −8.3 | 57.0± 0.1 | 11.1 | −8.8 | 55.4± 0.3 | 9.5 | −7.9 | 47.7± 0.2 | 1.8 | −9.2 |
| T103A | 46.8± 0.1 | 57.8± 0.2 | 11.0 | −5.7 | 60.6± 0.3 | 13.8 | −3.2 | 62.9± 0.2 | 16.1 | −4.7 | 64.9± 0.2 | 18.1 | −4.0 | 62.6± 0.3 | 15.8 | −4.6 | 53.3± 0.2 | 6.5 | −5.0 | 61.1± 0.3 | 14.3 | −5.6 | 59.7± 0.3 | 12.9 | −4.5 | 53.0± 0.2 | 6.2 | −4.8 |
| R104A | 40.9± 0.2 | 60.6± 0.4 | 19.7 | 3.0 | 60.1± 0.5 | 19.2 | 2.2 | 60.9± 0.3 | 20.0 | −0.8 | 63.1± 0.3 | 22.2 | 0.1 | 61.2± 0.3 | 20.3 | −0.1 | 52.0± 0.7 | 11.1 | −0.4 | 63.4± 0.3 | 22.5 | 2.6 | 60.4± 0.3 | 19.5 | 2.1 | 53.2± 0.4 | 12.3 | 1.3 |
| Q116A | n.d. | 54.4± 0.2 | n.d. | n.d. | 54.9± 0.1 | n.d. | n.d. | 59.4± 0.2 | n.d. | n.d. | 60.2± 0.1 | n.d. | n.d. | 59.0± 0.1 | n.d. | n.d. | 49.8± 0.4 | n.d. | n.d. | 57.1± 0.2 | n.d. | n.d. | 54.9± 0.3 | n.d. | n.d. | 44.1± 0.3 | n.d. | n.d. |
| R135A | 41.7± 0.2 | 62.2± 0.2 | 20.5 | 3.8 | 62.7± 0.1 | 21.0 | 4.0 | 70.1± 0.2 | 28.4 | 7.6 | 68.8± 0.1 | 27.1 | 5.0 | 70.0± 0.2 | 28.3 | 7.9 | 57.2± 0.1 | 15.5 | 4.0 | 67.9± 0.2 | 26.2 | 6.3 | 65.8± 0.2 | 24.1 | 6.7 | 56.9± 0.1 | 15.2 | 4.2 |
| S138A | 42.9± 0.1 | 59.7± 0.3 | 16.8 | 0.1 | 60.8± 0.1 | 17.9 | 0.9 | 63.4± 0.3 | 20.5 | −0.3 | 65.7± 0.2 | 22.8 | 0.7 | 62.9± 0.2 | 20.0 | −0.4 | 53.2± 0.1 | 10.3 | −1.2 | 62.3± 0.3 | 19.4 | −0.5 | 60.1± 0.3 | 17.2 | −0.2 | 53.5± 0.4 | 10.6 | −0.4 |
| E147A | 44.8 ± 0.1 | 56.6 ± 0.2 | 11.8 | −4.9 | 58.3± 0.1 | 13.5 | −3.5 | 59.2± 0.1 | 14.4 | −6.4 | 64.4± 0.1 | 19.6 | −2.5 | 59.5± 0.1 | 14.7 | −5.7 | 48.3± 0.2 | 3.5 | −8.0 | 55.9± 0.2 | 11.1 | −8.8 | 53.7± 0.2 | 8.9 | −8.5 | 49.0± 0.4 | 4.2 | −6.8 |
| R104A_ R135A* | n.d. | 56.1± 0.2 | n.d. | n.d. | 54.4± 0.2 | n.d. | n.d. | 55.5± 0.2 | n.d. | n.d. | 57.7± 0.4 | n.d. | n.d. | 57.0± 0.2 | n.d. | n.d. | n.d. | n.d. | n.d. | 55.5± 0.2 | n.d. | n.d. | 54.1± 0.1 | n.d. | n.d. | 53.9± 0.2 | n.d. | n.d. |
The indicated melting temperature (Tm) were derived using thermal shift assays. Apo, Unliganded; Chl, chlortetracycline; Dem, demeclocycline; Dox, doxycycline; Mec, meclocycline; Met, methacycline; Min, minocycline; Oxy, oxytetracycline; Tet, tetracycline; Tig, tigecycline. Experiments were repeated five times (technical repeats) except for * = three technical repeats. Mean Tm values and standard errors are shown. n.d. = not detected. ΔTm = Tm(liganded) – Tm(unliganded). ΔΔTm = ΔTm(Variant) – ΔTm(Wildtype). The numbers represent in °C as unit. Values in boldface represent Tm values of the substrate preferences of the respective AbTetR variants.