Structural Analysis of Kinetic Folding Intermediates for a TIM Barrel Protein, Indole-3-glycerol Phosphate Synthase, by Hydrogen Exchange Mass Spectrometry and Gō Model Simulation

• Published in: Journal of molecular biology Vol. 374; no. 2; pp. 528 - 546
• Main Authors: Gu, Zhenyu, Rao, Maithreyi K., Forsyth, William R., Finke, John M., Matthews, C. Robert
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 23.11.2007
• Subjects: Amino Acid Sequence; Computer Simulation; Deuterium Exchange Measurement; Hydrogen - chemistry; Indole-3-Glycerol-Phosphate Synthase - chemistry; kinetic folding mechanism; Kinetics; Mass Spectrometry; misfolded intermediate; Models, Molecular; Molecular Sequence Data; Protein Folding; stable intermediate; Sulfolobus solfataricus; topology; IOLI; sIGPS; I bp; MALDI; MS; topology; D 4-urea; N; TFA; kinetic folding mechanism; stable intermediate; U; I a and I b; TIM; protein folding; HD; HX-MS; αTS; misfolded intermediate
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2007.09.024

The structures of partially folded states appearing during the folding of a (βα) 8 TIM barrel protein, the indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (sIGPS), was assessed by hydrogen exchange mass spectrometry (HX-MS) and Gō 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 (βα) 4 region, modest protection in the neighboring (βα) 1–3 and (βα) 5β 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 (βα) 2–5β 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 α Gō 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 Gō 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 Gō model simulation.