Table 2.
Steady-state ATP hydrolysis rates at temperatures and buffer conditions of assay specified (i.e., EPR is under EPR buffer and temperature conditions). If not noted (top four reactions), conditions are the same as reference (Lavery et al., 2014)
| Protein | zTRAP1 ATPase (min−1) | hTRAP1 ATPase (min−1) |
|---|---|---|
| WT (30°C) | 1.36 ± 0.12 | 0.463 ± 0.003 |
| salt bridge point mutants (30°C) (Lavery et al., 2014) | (E-A) 3.57 ± 0.62 (H-A) 5.08 ± 0.90 | |
| Δstrap (30°C) | 5.84 ± 0.47 | 13.3 ± 0.5 |
| Δ60-69 (30°C) | 0.47 ± 0.02 | |
| WT FRET (30°C) | 0.21 ± 0.0.01 | |
| Δstrap FRET (30°C) | 11.9 ± 2.1 | |
| CFree WT EPR (23°C) | 0.88 ± 0.05 | |
| CFree Δstrap EPR (23°C) | 5.65 ± 0.22 | |
| CFree WT (Inter FRET) (30°C) | 0.79 ± 0.0.03 | |
| CFree Δstrap (Inter FRET) (30oC) | 7.6 ± 0.36 | |
| CFree WT (Intra FRET) (30°C) | 0.35 ± 0.0.01 |
Red text indicates WT or strap truncated protein with native cysteine and label free, while Blue indicates labeled protein used in FRET and EPR experiments (each in indicated buffer conditions). EPR samples are cysteine free except for the desired probe position and are spin-labeled. Inter FRET and Intra FRET samples are cysteine free except for the desired probe position and are labeled with Alexa Fluor dyes (Life Technologies, see ‘Materials and methods’). Note that ‘Intra FRET’ refers to both probe positions on the same promoter, whereas ‘Inter FRET’ refers to one probe position per promoter. Errors represent the standard deviation of triplicate experiments.