TABLE 4.
Sedimentation equilibrium analysis of the binding of FakB2 and its mutants to FakA
A reversible two-site heterogeneous association model was used for the analysis of the dissociation constants and the two discrete species model was used to determine the molar mass of the complex. CI is confidence interval; r.m.s.d. is root mean square deviation.
| Sample | μma | Kd, 1b (μm) (68.3% CI) | Kd, 2b (μm) (68.3% CI) | r.m.s.d.c | M1d | M2d | r.m.s.d.e |
|---|---|---|---|---|---|---|---|
| Da | Da | ||||||
| FakB2 | 15.75 | 1.71 | 6.84 | 0.0032 | 32,808 | 191,582 | 0.0028 |
| + FakA | + 6.54 | (1.64–11.06) | (6.5–44.26) | ||||
| FakB2(T61A) | 13.64 | 2.63 | 10.54 | 0.0026 | 32,778 | 179,621 | 0.0018 |
| + FakA | + 6.83 | (2.55–3.84) | (10.21–15.38) | ||||
| FakB2(S93A) | 11.86 | 1.46 | 5.87 | 0.0038 | 32,792 | 187,959 | 0.0037 |
| + FakA | + 6.54 | (1.42–3.74) | (5.70–14.96) | ||||
| FakB2(R170A) | 16.41 | 2.25 | 9.01 | 0.0035 | 32,723 | 181,313 | 0.0034 |
| + FakA | + 6.60 | (0.76–3.45) | (3.07–13.81) | ||||
| FakB2(R202A) | 24.80 + 5.39 | Not detected | Not detected | 32,723 | 132,268 | 0.0027 | |
| + FakA | |||||||
| FakB2(H226A) | 16.33 | 1.50 | 6.02 | 0.0033 | 32,742 | 187,964 | 0.0033 |
| + FakA | + 6.67 | (1.47–1.99) | (5.90–7.95) |
a Total loading concentrations in μm of FakB2 and its mutants (left-hand side) and FakA on the right in 20 mm Tris, pH 7.5, 0.2 m NaCl buffer at 4 °C.
b Dissociation equilibrium constants Kd, 1 for first site and Kd, 2 for the second site converted from the association equilibrium constants Ka, 1 and Ka, 2, respectively, for the reversible two-site heterogeneous association model with the association scheme A + B2 ↔ AB2 + B2 ↔ A(B2)2. FakA dimer (125,348 Da) was treated in the analysis as the single species A and FakB2 and mutants as B2.
c Root mean square deviation of the fit, units in absorbance for the reversible complex formations. Errors represent the 68.3% (one standard deviation) confidence interval (CI) using an automated surface projection method (33).
d Fitted molar masses of two discrete species with the first mass (M1) the theoretical molar mass of FakB2 and the mass of the second species (M2) floated in the fit.
e Root mean square deviation of the fit, units in absorbance for the two discrete species model. is shown.