Table 1.
DSC dataa and dimer dissociation constants (Kd) for selected PR constructs
| (A) | |||
|---|---|---|---|
| Protein | Tm (−DRV) | Tm (+DRV) | ΔTm (I)b |
| PR | 65.7c | 88.1c | 22.4 |
| PRL33F/E34D/L63P | nd | 85.4 | -- |
| PR20d | 71.7 | 77.0 | 5.3 |
| ANAM-11d | 70.5 | 81.9 | 11.4 |
| PRT26A | 60.0 | 63.0 | 3.0 |
| PR20T26A | 67.5 | 67.5 | 0 |
| PRT26A/L33F/L63P | 68.1 | 67.1 | −1.0 |
| (B) | |||||
|---|---|---|---|---|---|
| Protein | T m | T m | ΔTm (I)b | ΔTm (Sc)e | Kd (μM) |
| (− RPB) | (+ RPB) | ||||
| PRf | 56.6 | 65.8 | 9.2 | 11 (− RPB) | <0.01c |
| ScPRf | 67.6 | 75.4 | 7.8 | 9.6 (+ RPB) | -- |
| (− DRV) | (+ DRV) | ||||
| PRD25N | 58.4c | 61.5c | 3.1 | 9.0 (− DRV) | 1.3c |
| ScPRD25N | 67.4 | 70.3 | 2.9 | 8.8 (+ DRV) | -- |
a
At pH 4.7-5.0 in sodium acetate buffer unless noted otherwise
b
Tm(+inhibitor)-Tm(−inhibitor)
c
cited from reference21; an approximately 100-fold increase in Kd observed based on the active site mutation D25N in PR accompanies ~106-fold weaker binding of DRV
d
reference9
e
Tm(single chain)-Tm(homodimer)
f
in 25 mM sodium formate, pH 3.6
nd (not determined) due to more rapid autoproteolysis than PR, see Figure 2