Independent folding of individual components in hybrid proteins. Evidence that the carboxy-terminal 135 residues of the LexA repressor constitute a single autonomous domain.

Inactivation of the Escherichia coli repressor protein, LexA, takes place through a cleavage reaction which hydrolyzes the Ala84-Gly85 peptide bond near the center of the molecule. The mechanism of cleavage has previously been shown to be an intramolecular reaction stimulated in vitro by elevated pH or by the addition of activated RecA protein. The entire self-cleavage activity of LexA has been found to lie within a 135-residue tryptic fragment extending from Leu68 to the end of the protein at Leu202. Since the activity of self-cleavage is dependent on the proper three-dimensional structure of the protein, we have used it as a probe to investigate the extend of folding autonomy and functional independence of this 135-residue carboxy-terminal domain of LexA by applying a protein fusion approach. A series of twelve different hybrid proteins, containing LexA sequences in a variety of predefined primary structural arrangements, were constructed and evaluated for whether or not self-cleavage activity has been retained. The results revealed that retention or loss of activity is independent of the nature or size of the foreign protein used. Loss of self-cleavage was found to be a function of amino- or carboxy-terminal deletions in the self-cleaving LexA component of the fusion proteins. The present findings, together with the observations of other artificial fusions proteins and the naturally occurring bifunctional and multifunctional proteins, along with the data on helix packing, provide further support for the notion of modular architecture of proteins and suggest that when these autonomous units are fused, they retain their tendency to fold independently of the remainder of the polypeptide to generate physically linked active domains, rather than to fold dependently and yield scrambled structures.