TABLE 1.
Summary of rate and equilibrium constants for W127CLM, W127C, and mBBr-labeled W127C
Conditions were as follows: 25 mm HEPES, 50 mm potassium acetate, 5 mm magnesium acetate, 1 mm EGTA, 1 mm DTT, pH 7.50, 20 °C.
| Description | Methoda | W127CLM | W127C | mBBr-W127C |
|---|---|---|---|---|
| ATP-induced MT dissociation | Turbidity | 10.8 ± 1.3 | 10.1 ± 0.5 | 10.5 ± 0.3 |
| ATP induced mBBr quenching (s−1) | mBBr | 10.7 ± 1.3 | ||
| ADP induced mBBr quenching (s−1) | mBBr | 10.2 ± 2.1 | ||
| Eg5-ADP binding to MT (μm−1s−1) | Turbidity | 5.1 ± 0.9 | 4.3 ± 1.2 | 3.3 ± 1.1 |
| MT-activated ADP dissociation (s−1) | 2′dmD | 76 ± 15b | 5.5 ± 2.1 | |
| Weak → strong transition (s−1) | mBBr | 13.5 ± 1.2 | ||
| Steady state ATPase kcat (s−1) | 8.3 ± 0.5c | 4.4 ± 0.2 | 6.8 ± 0.2 | |
| Steady state ATPase K0.5,MT (μm) | 1.1 ± 0.2c | 0.24 ± 0.06 | 0.21 ± 0.05 |
a Methods include the following: 1) 2′dmD, FRET from microtubule tryptophans to 2′dmD fluorophore; 2) mBBr, fluorescence change in monobromobimane probe at position 127; 3) turbidity, measured in the stopped flow spectrophotometer at 350 nm (W127CLM and W127C) or 420 nm (mBBr-labeled W127C).
b Data from Ref. 4.
c Measurement of ATP-induced dissociation was accomplished by mixing the MD·MT complex with ATP in 200 mm KCl to induce complete dissociation.