Table 2.
Protein | Nonlinear fitting |
AUC analysis |
|||
---|---|---|---|---|---|
Kd (μM)∗ | No substrate |
With 600 μM substrate |
|||
Kd (μM)† | koff (s−1)† | Kd (μM)† | koff (s−1)† | ||
Wild-type | 0.8 ± 0.4 | 2.0 ± 0.01 | 0.1 ± 0.007 | 1.7 ± 0.03 | 0.1 ± 0.002 |
R298A | 11.4 ± 9.7 | 81.1 ± 3.3 | 0.1 ± 0.004 | 4.7 ± 0.1 | 0.08 ± 0.001 |
R298L | 13.8 ± 8.7 | 30.8 ± 0.4 | 0.1 ± 0.001 | 2.6 ± 0.1 | 0.1 ± 0.003 |
R298A/Q299A | NDc | 85.7 ± 3.1 | 0.1 ± 0.004 | 115.0 ± 4.4 | 0.05 ± 0.002 |
E166A | ND | 3.7 ± 0.2 | 0.04 ± 0.001 | 0.4 ± 0.01 | 0.1 ± 0.002 |
E166A/R298A | ND | 141.1 ± 12.7 | 0.1 ± 0.009 | 103.7 ± 8.3 | 0.06 ± 0.005 |
Kd was calculated by nonlinear dependence equation (Eq. 3).
Parameters were derived from a global fit of the SV and SE data to a monomer-dimer self-association model by SEDPHAT. The SE experiments for the assay were obtained at protein concentrations of 5.7–28.6 μM, whereas SV experiments were at 1.4–57.2 μM.