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 |
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Authors: | Forsyth William R Bilsel Osman Gu Zhenyu Matthews C Robert |
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Institution: | Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA, 01605, USA. |
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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. |
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Keywords: | αTS α-subunit of tryptophan synthase from Escherichia coli ANS 1-anilino-8-naphthalene sulfonate FL fluorescence I1 equilibrium intermediate of αTS populated in 3 M urea I2 equilibrium intermediate of αTS populated in 5 M urea Ibp kinetic intermediate populated within the stopped-flow burst phase (< 10 ms) IOLI the 278 residue TIM barrel protein of unknown function encoded by the Bacillus subtilis iolI gene MRE mean residue ellipticity N native state Rh hydrodynamic radius sIGPS indole-glycerol-phosphate synthase from Sulfolobus solfataricus U unfolded state |
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