A number of studies have examined the structural properties of late folding intermediates of (β/α)8-barrel proteins involved in tryptophan biosynthesis, whereas there is little information available about the early folding events of these proteins. To identify the contiguous polypeptide segments important to the folding of the (β/α)8-barrel protein Escherichia coli N-(5′-phosphoribosyl)anthranilate isomerase, we structurally characterized fragments and circularly permuted forms of the protein. We also simulated thermal unfolding of the protein using molecular dynamics. Our fragmentation experiments demonstrate that the isolated (β/α)1-4β5 fragment is almost as stable as the full-length protein. The far and near-UV CD spectra of this fragment are indicative of native-like secondary and tertiary structures. Structural analysis of the circularly permutated proteins shows that if the protein is cleaved within the two N-terminal βα modules, the amount of secondary structure is unaffected, whereas, when cleaved within the central (β/α)3-4β5 segment, the protein simply cannot fold. An ensemble of the denatured structures produced by thermal unfolding simulations contains a persistent local structure comprised of β3, β4 and β5. The presence of this three-stranded β-barrel suggests that it may be an important early-stage folding intermediate. Interactions found in (β/α) 3-4β5 may be essential for the early events of ePRAI folding if they provide a nucleation site that directs folding.
ASJC Scopus subject areas