An 8-fold beta alpha barrel protein with redundant folding possibilities.

Protein sequences containing redundant segments of secondary structure at both termini have the choice a priori of folding into several possible circularly permuted variants of the wild-type tertiary structure. To test this hypothesis the gene of phosphoribosyl anthranilate isomerase from yeast, which is a single-domain 8-fold beta alpha barrel protein, was modified to produce a 10-fold beta alpha homologue in Escherichia coli. It contained a duplicate of the two C-terminal beta alpha units of supersecondary structure fused to its N-terminus. Most of the protein was recovered from the insoluble fraction of disrupted cells by dissolution in guanidinium chloride solutions and refolding. Pristine protein was purified from the soluble fraction. The purified (beta alpha)10 proteins were enzymically almost fully active. Absorbance, fluorescence and circular dichroism spectra as well as the reversible unfolding behaviour of both proteins were also very similar to the properties of the original (beta alpha)8 protein. Digestion with endopeptidases converted both the pristine and the refolded (beta alpha)10 variant to the same large fragment that had the N-terminal sequence and mol. wt of the wild-type (beta alpha)8 protein. The data suggest that the folding of the (beta alpha)10 variant is controlled thermodynamically both in vivo and in vitro.