title: 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.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA, 01605, USA.
authors.forename: William R
authors.initials: WR
authors.lastname: Forsyth
authors.forename: Osman
authors.initials: O
authors.lastname: Bilsel
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Gu
authors.forename: C Robert
authors.initials: CR
authors.lastname: Matthews
mesh_terms: D000595:Amino Acid Sequence; D016880:Anisotropy; D001412:Bacillus subtilis / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D001426:Bacterial Proteins