Table 1.
Experimental determination of cis- or trans-topology of several NBD-labeled mutants using fluorescence lifetime quenching with LysoUB (22). τi and αi are individual lifetime components; τα and τ0 are amplitude-averaged lifetimes in the presence and in the absence of LysoUB quencher. Cis and Trans topologies are assigned based on the value of τα / τ0 ratio based on the hallmarks established in a model study (22).
| T-Domain labeling site |
LUV | LysoUB | τ1, ns(α1) | τ2, ns(α2) | τα, ns | τα/τ0 (Topology) |
|---|---|---|---|---|---|---|
| 350C-NBD | 25PC:75PG | None + 2% |
1.50 (0.32) 1.42 (0.37) |
7.90 (0.68) 7.47 (0.63) |
5.85 5.25 |
1.12 (Trans) |
| 350C-NBD | 75PC:25PG | None + 2% |
1 33 (0.35) 1.32 (0.40) |
7.20 (0.65) 6.79 (0.60) |
5.13 4.61 |
1.11 (Trans) |
| 350C-NBD | 90PC:10PG | None + 2% |
1 18 (0.50) 0.94 (0.59) |
6.27 (0.50) 3.97 (0.41) |
3.72 2.19 |
1.70 (Cis) |
| 378C-NBD | 25PC:75PG | None + 2% |
1.23 (0.51) 0.85 (0.67) |
4.54 (0.49) 3.50 (0.33) |
2.98 1.73 |
1.72 (Cis) |