A SINGLE AMINO-ACID SUBSTITUTION CAN RESTORE THE SOLUBILITY OF AGGREGATED COLICIN-A MUTANTS IN ESCHERICHIA-COLI

Mutants of colicin A have been prepared in which the three tryptophan residues (Trp86, Trp130 acid Trp140), localized in the C-terminal doma in of the soluble wildtype protein, have been substituted by phenylala nine. The Trp140Phe single mutation had the effect of decreasing the p ercentage of protein that is expressed as insoluble aggregates. The cr eated hydrophobic cavity decreased the stability of the protein during its folding, resulting in partial aggregation in the cytoplasm of the Escherichia coli-producing cells. Aggregation was increased when Trp1 40 was substituted by Lys, Leu or Cys, or if the Trp140 mutation was c ombined with the Trp86Phe and/or Trp130Phe mutations. A single mutatio n, Lys113Phe, however, was able to restore the solubility of the aggre gated mutants in vivo. Detailed analysis of the 3-D structure of the C -terminal domain of colicin A suggests that filling of the hydrophobic cavity is responsible for this effect.