Random circular permutation of DsbA reveals segments that are essential for protein folding and stability

One of the key questions in protein folding is whether polypeptide chains r equire unique nucleation sites to fold to the native state, In order to ide ntify possible essential polypeptide segments for folding, we have performe d a complete circular permutation analysis of a protein in which the natura l termini are in close proximity. As a model system, we used the disulfide oxidoreductase DsbA from Escherichia coli, a monomeric protein of 189 amino acid residues. To introduce new termini at all possible positions in its p olypeptide chain, we generated a library of randomly circularly permuted ds bA genes and screened for active circularly permuted variants in vivo. A to tal of 51 different active variants were identified. The new termini were d istributed over about 70 % of the poly peptide chain, with the majority of them occurring within regular secondary structures. New termini were not fo und in approximately 30 % of the DsbA sequence which essentially correspond to four alpha-helices of DsbA. Introduction of new termini into these "for bidden segments" by directed mutagenesis yielded proteins with altered over all folds and strongly reduced catalytic activities. In contrast, all activ e variants analysed so far show structural and catalytic properties compara ble with those of DsbA wild-type. We suggest that random circular permutati on allows identification of contiguous structural elements in a protein tha t are essential for folding and stability. (C) 1999 Academic Press.