Table 2. Characterization of the DN mutants.
| Mutant | LFN binding | LFN translocation | Pore formation | Heptamer formation | SDS-resistant heptamer |
|---|---|---|---|---|---|
| WT | +++ | +++ | +++ | +++ | +++ |
| I364C | +++ | - | - | +++ | - |
| T380C | +++ | - | - | +++ | - |
| S382C | +++ | - | - | +++ | - |
| T393C | +++ | - | - | +++ | - |
| N399C | +++ | - | - | +++ | - |
| Y411C | +++ | - | - | +++ | + |
| N422C | +++ | - | - | +++ | - |
| D425K* | +++ | - | - | +++ | - |
| K397D* | +++ | - | - | +++ | - |
| F427A* | +++ | - | - | +++ | +++ |
All of the mutants tested showed <0.01 wild-type PA activity in mediating the toxicity of LFNDTA. LFN binding is measured as the amount of LFN bound to CHO cells preincubated with trypsin-nicked PA (nPA). LFN translocation is the percentage of bound LFN protected from proteolysis after a pulse at pH 5. Pore formation is measured as the amount of 86Rb+ released from preloaded cells incubated with nPA and pulsed at pH 5. Heptamer formation is visualized by native PAGE of equimolar mixes of nPA with LFN. Formation of SDS-resistant heptamers is triggered by dropping the pH of the mixes to pH 5 and is visualized by SDS/PAGE. The activities are reported as +++, 75-100%; ++, 25-74%; +, 5-24%, and -, <5% of wild-type levels.
The data for these mutants are derived from previous experiments (41).