Topology and Sequence in the Folding of a TIM Barrel Protein: Global Analysis Highlights Partitioning between Transient Off-pathway and Stable On-pathway Folding Intermediates in the Complex Folding Mechanism of a (βα)8 Barrel of Unknown Function from B.

The relative contributions of chain topology and amino acid sequence in directing the folding of a (βα)8 TIM barrel protein of unknown function encoded by the Bacillus subtilis iolI gene (IOLI) were assessed by reversible urea denaturation and a combination of circular dichroism, fluorescence and time-resolved fluorescence anisotropy spectroscopy. The equilibrium reaction for IOLI involves, in addition to the native and unfolded species, a stable intermediate with significant secondary structure and stability and self-associated forms of both the native and intermediate states. Global kinetic analysis revealed that the unfolded state partitions between an off-pathway refolding intermediate and the on-pathway equilibrium intermediate early in folding. Comparisons with the folding mechanisms of two other TIM barrel proteins, indole-3-glycerol phosphate synthase from the thermophile Sulfolobus solfataricus (sIGPS) and the alpha subunit of Escherichia coli tryptophan synthase (αTS), reveal striking similarities that argue for a dominant role of the topology in both early and late events in folding. Sequence-specific effects are apparent in the magnitudes of the relaxation times and relative stabilities, in the presence of additional monomeric folding intermediates for αTS and sIGPS and in rate-limiting proline isomerization reactions for αTS.