Table 1. Quantification of Binding Affinity and Thermal Stability of WT and Mutant VHH Domainsa.
| ITC |
SPR |
DSF |
||||||
|---|---|---|---|---|---|---|---|---|
| sample | ΔH (cal·mol–1) | ΔS (cal·mol–1·deg–1) | KD (nM) | koff (10–3 s–1) | kon (103 M–1 s–1) | KD (nM) | Tm1 (°C) | Tm2 (°C) |
| VHH(WT) | –5187 | 15.4 | 68.97 ± 12.70 | 8.53 ± 0.04 | 52.34 ± 15.4 | 162.97 ± 47.53 | 52.35 ± 0.04 | 70.2 ± 0.14 |
| C24A | –4691 | 16.6 | 86.96 ± 9.81 | 10.67 ± 0.04 | 55.25 ± 17.91 | 193.12 ± 61.18 | 47.25 ± 0.04 | ND |
| C98A | –4318 | 17.2 | 121.80 ± 17.36 | 10.82 ± 0.04 | 55.82 ± 9.98 | 193.84 ± 31.38 | 46.80 ± 0.10 | ND |
| C24A/C98A | –4120 | 17.2 | 168.92 ± 31.66 | 11.55 ± 0.06 | 54.70 ± 19.30 | 211.15 ± 85.04 | 47.02 ± 0.06 | ND |
a
ND, not detected; errors indicate 95% confidence intervals (see respective methods in Supporting Information).