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 tryptophanresidues (Trp86, Trpl30 and Trpl40), localized in the C-terminaldomain of the soluble wild-type protein, have been substitutedby phenylalanine. The Trpl40Phe single mutation had the effectof decreasing the percentage of protein that is expressed asinsoluble aggregates. The created hydrophobic cavity decreasedthe stability of the protein during its folding, resulting inpartial aggregation in the cytoplasm of the Escherichia coli-producingcells. Aggregation was increased when Trpl40 was substitutedby Lys, Leu or Cys, or if the Trpl40 mutation was combined withthe Trp86Phe and/ or Trpl30Phe mutations. A single mutation,Lysll3Phe, however, was able to restore the solubility of theaggregated mutants in vivo. Detailed analysis of the 3-D structureof the C-terminal domain of colicin A suggests that fillingof the hydrophobic cavity is responsible for this effect.