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.
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 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 (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) 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.
Gu
Zhenyu
Z
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Rao
Maithreyi K
MK
Forsyth
William R
WR
Finke
John M
JM
Matthews
C Robert
CR
D000595:Amino Acid Sequence; D003198:Computer Simulation; D041961:Deuterium Exchange Measurement; D006859:Hydrogen / Q000737:chemistry*; D007209:Indole-3-Glycerol-Phosphate Synthase / Q000737:chemistry*; D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017510:Protein Folding
D006859:Hydrogen; D007209:Indole-3-Glycerol-Phosphate Synthase