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 βα barrel protein, was modified to produce a 10-fold βα homologue in Escherichia coli. It contained a duplicate of the two C-terminal βα 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 (βα)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 (βα)8 protein. Digestion with endopeptidases converted both the pristine and the refolded (βα)10 variant to the same large fragment that had the N-terminal sequence and mol. wt of the wild-type βα)8 protein. The data suggest that the folding of the (βα)10 variant is controlled thermodynamically both in vivo and in vitro. © 1990 Oxford University Press.