Mapping the Structure of Folding Cores in TIM Barrel Proteins by Hydrogen Exchange Mass Spectrometry: The Roles of Motif and Sequence for the Indole-3-glycerol Phosphate Synthase from Sulfolobus solfataricus

Abstract

To test the roles of motif and amino acid sequence in the folding mechanisms of TIM barrel proteins, hydrogen-deuterium exchange was used to explore the structure of the stable folding intermediate for the of indole-3-glycerol phosphate synthase from S. solfataricus (sIGPS). Previous circular dichroism and fluorescence studies of the urea denaturation of sIGPS revealed the presence of an intermediate that is highly populated at ∼4.5 M urea and contains ∼50% of the secondary structure of the native state. Kinetic studies showed that this apparent equilibrium intermediate is actually comprised of two thermodynamically-distinct species, I a and I b . To probe the location of the secondary structure in this pair of stable on-pathway intermediates, the equilibrium unfolding process of sIGPS was monitored by hydrogen-deuterium (HD) exchange mass spectrometry. The intact protein and pepsin-digested fragments were studied at various urea concentrations by electrospray and MALDI-TOF mass spectrometry, respectively, after deuterium labeling and quenching in acid. Intact sIGPS strongly protects 54 amide hydrogens from HD exchange in the intermediate states, demonstrating the presence of stable folded cores. When the protection patterns and the exchange mechanisms for the peptides are considered with the proposed folding mechanism, the results can be interpreted to define the structural boundaries of I a and I b . Comparison of these results with previous HD exchange studies on another TIM barrel protein of low sequence identify, the alpha subunit of tryptophan synthase (αTS), indicates that the thermodynamic states corresponding to the folding intermediates are better conserved than their structures. Although the TIM barrel motif appears to define the basic features of the folding free energy surface, the structures of the partiallyfolded states that appear during the folding reaction depend on the amino acid sequence. Markedly, the good correlation between the HD exchange patterns of sIGPS and αTS with the locations of hydrophobic clusters defined by isoleucines, leucines, and valines suggests that branch aliphatic side chains play a critical role in defining the structures of the equilibrium intermediates.

Repeat-protein folding: New insights into origins of cooperativity, stability, and topology

“…In a few cases where hydrogen exchange and mutagenesis studies have begun to provide a measure of the energy distribution for globular proteins, a relationship has been found between local stability imbalance and the formation of equilibrium folding intermediates [71], and substitutions magnifying this imbalance promote multistate unfolding [53].…”

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

“…25,26 However, the deletion of the first 26 residues, containing the α00 α-helix, results in a fully-reversible unfolding reaction that enabled a previous HX analysis of the structures of its equilibrium intermediates. 24 This truncated form of sIGSP also served as the target of the present study of its sub-millisecond folding reaction.…”

“…5 A third type of response is displayed by IOLI, whose unfolded form is thought to partition between off-and on-pathway intermediates in the millisecond time frame. 22 Previous HX analyses of the equilibrium folding intermediates in αTS 23 and sIGPS, 24 employing pepsin digestion at acidic pH where mass-labeled amides can be examined by MALDI mass spectrometry, provided insights into the structures of their respective partiallyfolded forms. In the case of sIGPS, the HX-MS data were sufficiently rich to allow the determination of the structures of a pair of stable intermediates predicted by kinetic studies of the folding reaction.…”

“…5 Differences in the structures of these equilibrium intermediates were attributed to sequence-specific hydrophobic clusters formed by isoleucine, leucine and valine side chains. 24 Complications with the folding properties of αTS, i.e., four parallel folding channels, and IOLI, i.e., a weakly-folded and self-associating native state, led to the choice of sIGPS as the initial target of an HX-MS analysis of protection in its burst-phase species.…”

“…24 A re-examination of the kinetic folding mechanism revealed an off-pathway burst-phase intermediate whose unfolding under refolding conditions leads to the on-pathway intermediates. Complementary Gō-model simulations of a C α representation of sIGPS support the conclusion that the topology of a TIM barrel protein dictates its funnel-shaped folding free energy surface and the presence of the on-pathway intermediates.…”