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 (betaalpha)8 barrel of unknown function from B. subtilis

UMMS Affiliation

Department of Biochemistry and Molecular Pharmacology

Publication Date


Document Type



Amino Acid Sequence; Anisotropy; *Bacillus subtilis; Bacterial Proteins; Kinetics; Models, Molecular; Molecular Sequence Data; *Protein Folding; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Temperature


Biochemistry, Biophysics, and Structural Biology | Pharmacology, Toxicology and Environmental Health


The relative contributions of chain topology and amino acid sequence in directing the folding of a (betaalpha)(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 (alphaTS), 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 alphaTS and sIGPS and in rate-limiting proline isomerization reactions for alphaTS.

DOI of Published Version



J Mol Biol. 2007 Sep 7;372(1):236-53. Epub 2007 Jun 14. Link to article on publisher's site

Journal/Book/Conference Title

Journal of molecular biology

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Link to Article in PubMed

PubMed ID