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Structural analysis of kinetic folding intermediates for a TIM barrel protein, indole-3-glycerol phosphate synthase, by hydrogen exchange mass spectrometry and Go model simulation
Biochemistry and Molecular Pharmacology Publications and Presentations
  • Zhenyu Gu, University of Massachusetts Medical School
  • Maithreyi K. Rao, Oakland University
  • William R. Forsyth
  • John M. Finke, Oakland University
  • C. Robert Matthews, University of Massachusetts Medical School
UMMS Affiliation
Department of Biochemistry and Molecular Pharmacology
Date
10-19-2007
Document Type
Article
Medical Subject Headings
Amino Acid Sequence; *Computer Simulation; *Deuterium Exchange Measurement; Hydrogen; Indole-3-Glycerol-Phosphate Synthase; Kinetics; Mass Spectrometry; Models, Molecular; Molecular Sequence Data; *Protein Folding
Abstract
The structures of partially folded states appearing during the folding of a (betaalpha)(8) TIM barrel protein, the indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (sIGPS), was assessed by hydrogen exchange mass spectrometry (HX-MS) and Go model simulations. HX-MS analysis of the peptic peptides derived from the pulse-labeled product of the sub-millisecond folding reaction from the urea-denatured state revealed strong protection in the (betaalpha)(4) region, modest protection in the neighboring (betaalpha)(1-3) and (betaalpha)(5)beta(6) segments and no significant protection in the remaining N and C-terminal segments. These results demonstrate that this species is not a collapsed form of the unfolded state under native-favoring conditions nor is it the native state formed via fast-track folding. However, the striking contrast of these results with the strong protection observed in the (betaalpha)(2-5)beta(6) region after 5 s of folding demonstrates that these species represent kinetically distinct folding intermediates that are not identical as previously thought. A re-examination of the kinetic folding mechanism by chevron analysis of fluorescence data confirmed distinct roles for these two species: the burst-phase intermediate is predicted to be a misfolded, off-pathway intermediate, while the subsequent 5 s intermediate corresponds to an on-pathway equilibrium intermediate. Comparison with the predictions using a C(alpha) Go model simulation of the kinetic folding reaction for sIGPS shows good agreement with the core of the structure offering protection against exchange in the on-pathway intermediate(s). Because the native-centric Go model simulations do not explicitly include sequence-specific information, the simulation results support the hypothesis that the topology of TIM barrel proteins is a primary determinant of the folding free energy surface for the productive folding reaction. The early misfolding reaction must involve aspects of non-native structure not detected by the Go model simulation.
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Citation: J Mol Biol. 2007 Nov 23;374(2):528-46. Epub 2007 Sep 14. Link to article on publisher's site
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Link to Article in PubMed
PubMed ID
17942114
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Citation Information
Zhenyu Gu, Maithreyi K. Rao, William R. Forsyth, John M. Finke, et al.. "Structural analysis of kinetic folding intermediates for a TIM barrel protein, indole-3-glycerol phosphate synthase, by hydrogen exchange mass spectrometry and Go model simulation" Vol. 374 Iss. 2 (2007) ISSN: 0022-2836 (Linking)
Available at: http://works.bepress.com/john_finke/11/