Table 2.
Site-directed mutagenesisa
| Mutated ColD residue | In vivob |
In vitroc |
Mutated ImmD residue | In vivob | In vitroc | |
|---|---|---|---|---|---|---|
| colD+ImmD | −ImmD | +ImmD | colD+ImmD | colD+ImmD | ||
| wt ColD | + | + | − | wt ImmD | + | − |
| L595P | + | + | − | K11A | + | − |
| S601P | + | + | − | L12A | + | − |
| R602G | + | + | − | S16A | + | − |
| K603E | + | + | − | K17A | + | − |
| D606H | + | + | − | E22A | + | − |
| K607A | +/−− | +/−− | − | R26A | + | − |
| K608A | − | − | ND | E30A | + | − |
| K610A | − | − | ND | R31A | + | + |
| H611A | − | − | ND | R32A | + | − |
| D614A | +/−− | + | +/− | R32Q | + | +/− |
| Y649A | +/− | +/− | − | R33A | + | − |
| R651A | − | − | ND | Y35A | + | − |
| E652A | + | + | − | N46A | + | +/− |
| K670A | + | + | − | E49A | + | +/−− |
| S677A | − | − | ND | F52A | + | + |
| W679A | − | − | ND | M53A | + | − |
| K680A | + | + | − | E56A | + | +/− |
| N682A | +/− | +/− | − | R57A | + | +/−− |
| A685Q | +/− | + | +/− | E59A | + | + |
| D686A | + | +/− | − | D61A | + | − |
| D63A | + | − | ||||
| Y67A | + | − | ||||
| E68A | + | − | ||||
| N72A | + | +/− | ||||
| K75A | + | + | ||||
| V76A | + | +/− | ||||
| R79A | + | + | ||||
| E83A | + | +/− | ||||
| |
|
|
|
K84A |
+ |
+/− |
| Substitutions of functionally important residues are indicated by bold characters. | ||||||
| bThe cytotoxicity of the mutant proteins, spotted directly onto a lawn of sensitive wild-type cells, was determined by halo test. | ||||||
| cThe tRNase catalytic activity was analyzed in vitro by Northern blot: (+) wild-type activity; (+/−)=about 50% of wild-type activity; (+/−−) weak activity; (−) no activity; (ND) not determined. No tRNase activity detected in the presence of ImmD protein, signifies a wild-type level of immunity exhibited by the colicin D–ImmD complex tested. | ||||||
Full-sized wild-type or mutated colicin D molecules, with or without the wild-type or mutated ImmD protein are synthesized in a coupled E. coli S30 transcription/translation system (see Materials and methods).