Multi-state Unfolding of the Alpha Subunit of Tryptophan Synthase, a TIM Barrel Protein: Insights into the Secondary Structure of the Stable Equilibrium Intermediates by Hydrogen Exchange Mass Spectrometry

Rojsajjakul, Teerapat; Wintrode, Patrick L.; Vadrevu, Ramakrishna; Matthews, C. Robert; Smith, David L.

doi:10.1016/j.jmb.2004.05.062

Cited by 35 publications

(50 citation statements)

References 38 publications

(42 reference statements)

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“…Although a Gō-model might be expected to eliminate intermediates and fold directly to the native conformation, variations in loop lengths between elements of secondary structure, variations in packing density, etc., might lead to differential stabilization of sub-domains and, thereby, folding intermediates. A previous Gō-model analysis of the folding mechanism of αTS 32 revealed partially-folded states that strongly resembled those identified by HX-MS analysis of peptic peptides 23 and native-state HX-NMR analysis of the full-length protein (Vadrevu, Wu and Matthews, manuscript in preparation).…”

Section: Gō-model Simulation Of Sigps Folding Mechanismmentioning

confidence: 62%

“…A previous comparison of the HX protection patterns offered by the I a and I b intermediates from sIGPS and the I1 intermediate from αTS 23,24 revealed that the secondary structures are not identical. The I1 species in αTS displays strong protection in the (βα) 1-4 segment, while the composite I a and I b species in sIGPS display strong protection in the β 2 (βα) 3-5 β 6 and the α 1 (βα) 2-7 segments, respectively.…”

Section: Barrel Proteinsmentioning

confidence: 94%

“…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.…”

Section: Introductionmentioning

confidence: 99%

“…This approach has previously been used to determine the relative percentage protection of peptides after HD exchange. 23,24,62 …”

mentioning

confidence: 99%

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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 Gō Model Simulation

Rao

Forsyth

et al. 2007

Journal of Molecular Biology

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The structures of partially-folded states appearing during the folding of a (βα) 8 TIM barrel protein, the indole-3-glycerol phosphate synthase from S. 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) 5 β 6 segments and no significant protection in the remaining N-and Cterminal 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) 2-5 β 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 structure offering protection against exchange in the on-pathway intermediate (s). Because the native-centric Gō-model simulations do not explicitly include sequencespecific 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.

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Section: Gō-model Simulation Of Sigps Folding Mechanismmentioning

confidence: 62%

Section: Barrel Proteinsmentioning

confidence: 94%

Section: Introductionmentioning

confidence: 99%

“…This approach has previously been used to determine the relative percentage protection of peptides after HD exchange. 23,24,62 …”

mentioning

confidence: 99%

See 2 more Smart Citations

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

Rao

Forsyth

et al. 2007

Journal of Molecular Biology

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“…Three-state folding occurs when a native-like intermediate additionally contains some slowly repaired misfolding error. In agreement, folding intermediates for many proteins are seen to be partial replicas of the native protein, whether they are kinetically populated or not, and the kinetically blocked forms are also seen to contain some significant misfolding (Kiefhaber et al 1992;Dobson et al 1994;Elöve et al 1994;Muñoz et al 1994;Sosnick et al 1994Sosnick et al , 1996Weissman and Kim 1995;Silow and Oliveberg 1997;Bai 1999;Bilsel et al 1999;Bhuyan and Udgaonkar 2001;Capaldi et al 2002;Wallace and Matthews 2002;Krishna et al 2003aKrishna et al , 2004Bollen et al 2004;Rojsajjakul et al 2004;Religa et al 2005;Wintrode et al 2005;Nishimura et al 2006).…”

Section: Three-state Foldingmentioning

confidence: 71%

A unified mechanism for protein folding: Predetermined pathways with optional errors

Mallela

Englander

2007

Protein Science

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There is a fundamental conflict between two different views of how proteins fold. Kinetic experiments and theoretical calculations are often interpreted in terms of different population fractions folding through different intermediates in independent unrelated pathways (IUP model). However, detailed structural information indicates that all of the protein population folds through a sequence of intermediates predetermined by the foldon substructure of the target protein and a sequential stabilization principle. These contrary views can be resolved by a predetermined pathway-optional error (PPOE) hypothesis. The hypothesis is that any pathway intermediate can incorporate a chance misfolding error that blocks folding and must be reversed for productive folding to continue. Different fractions of the protein population will then block at different steps, populate different intermediates, and fold at different rates, giving the appearance of multiple unrelated pathways. A test of the hypothesis matches the two models against extensive kinetic folding results for hen lysozyme which have been widely cited in support of independent parallel pathways. The PPOE model succeeds with fewer fitting constants. The fitted PPOE reaction scheme leads to known folding behavior, whereas the IUP properties are contradicted by experiment. The appearance of a conflict with multipath theoretical models seems to be due to their different focus, namely on multitrack microscopic behavior versus cooperative macroscopic behavior. The integration of three well-documented principles in the PPOE model (cooperative foldons, sequential stabilization, optional errors) provides a unifying explanation for how proteins fold and why they fold in that way.Keywords: protein folding; misfolding; foldons; sequential stabilization; optional error; predetermined pathway; lysozyme Detailed structural information obtained from hydrogen exchange (HX) studies of cytochrome c (Cyt c) and related studies with other proteins support a classical view of protein folding in which all of the molecules in a refolding population fold through essentially the same intermediate structures (Chamberlain and Marqusee 2000;Englander 2000;Maity et al. 2005). The pathway constructs the native protein by the stepwise addition of the cooperative folding units of the native structure, called foldons. The order of pathway steps is guided by a sequential stabilization process in which prior native-like structure templates the formation of subsequent complementary structure. Thus the folding pathway is determined by the same cooperative interactions that determine the target native structure. The pathway may be linear or may branch at any step as directed by the logic of the sequential stabilization process (Krishna et al. 2006 449quite differently, in terms of independent unrelated pathways (IUP model). Proteins can fold in a simple two-state way with only the unfolded (U) and native (N) states significantly populated, or in a multistate way with one or more intermediates (...

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RheoScale: A tool to aggregate and quantify experimentally determined substitution outcomes for multiple variants at individual protein positions

et al. 2018

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Human mutations often cause amino acid changes (variants) that can alter protein function or stability. Some variants fall at protein positions that experimentally exhibit "rheostatic" mutation outcomes (different amino acid substitutions lead to a range of functional outcomes). In ongoing studies of rheostat positions, we encountered the need to aggregate experimental results from multiple variants, to describe the overall roles of individual positions. Here, we present "RheoScale" which generates quantitative scores to discriminate rheostat positions from those with "toggle" (most substitutions abolish function) or "neutral" (most substitutions have wild-type function) outcomes. RheoScale scores facilitate correlations of experimental data (such as binding affinity or stability) with structural and bioinformatic analyses. The RheoScale calculator is encoded into a Microsoft Excel workbook and an R script. Example analyses are shown for three model protein systems, including one assessed via deep mutational scanning. The RheoScale calculator quickly and efficiently provided quantitative descriptions that were in good agreement with prior qualitative observations. As an example application, scores were compared to the example proteins' structures; strong rheostat positions tended to occur in dynamic locations. In the future, RheoScale scores can be easily integrated into computational studies to facilitate improved algorithms for predicting outcomes of human variants.

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Multi-state Unfolding of the Alpha Subunit of Tryptophan Synthase, a TIM Barrel Protein: Insights into the Secondary Structure of the Stable Equilibrium Intermediates by Hydrogen Exchange Mass Spectrometry

Cited by 35 publications

References 38 publications

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

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

A unified mechanism for protein folding: Predetermined pathways with optional errors

RheoScale: A tool to aggregate and quantify experimentally determined substitution outcomes for multiple variants at individual protein positions

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