Equilibrium and Kinetic Folding of Rabbit Muscle Triosephosphate Isomerase by Hydrogen Exchange Mass Spectrometry

Pan, Hai; Raza, Ashraf; Smith, David L.

doi:10.1016/j.jmb.2003.12.076

Cited by 39 publications

(52 citation statements)

References 31 publications

(34 reference statements)

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“…Hydrogen exchange mass spectrometry (HX-MS) has recently been used to obtain structural information on folding intermediates of several TIM barrel proteins, including αTS, 18 the multimeric aldolase 19 20 The results were interpreted in terms of a 4+4 Cterminus driven folding mechanism, similar to HisF. 16 Two rare partially-folded states in equilibrium with native dimeric yeast TIM were detected by a complementary technique, misincorporation proton-alkyl exchange (MPAX).…”

Section: Introductionmentioning

confidence: 99%

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

Zitzewitz

Matthews

2007

Journal of Molecular Biology

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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.

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Section: Introductionmentioning

confidence: 99%

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

Zitzewitz

Matthews

2007

Journal of Molecular Biology

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“…Again, early visual assessment found that triosephosphate isomerase "cannot be plausibly subdivided [into domains]" (16). Later experimental evidence, however, suggests that TIM barrels comprise two or even three domains (18,19). Clearly, a rigorous, quantitative domain definition is needed.…”

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confidence: 99%

A thermodynamic definition of protein domains

Porter

Rose

2012

Proc. Natl. Acad. Sci. U.S.A.

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Protein domains are conspicuous structural units in globular proteins, and their identification has been a topic of intense biochemical interest dating back to the earliest crystal structures. Numerous disparate domain identification algorithms have been proposed, all involving some combination of visual intuition and/or structurebased decomposition. Instead, we present a rigorous, thermodynamically-based approach that redefines domains as cooperative chain segments. In greater detail, most small proteins fold with high cooperativity, meaning that the equilibrium population is dominated by completely folded and completely unfolded molecules, with a negligible subpopulation of partially folded intermediates. Here, we redefine structural domains in thermodynamic terms as cooperative folding units, based on m-values, which measure the cooperativity of a protein or its substructures. In our analysis, a domain is equated to a contiguous segment of the folded protein whose mvalue is largely unaffected when that segment is excised from its parent structure. Defined in this way, a domain is a self-contained cooperative unit; i.e., its cooperativity depends primarily upon intrasegment interactions, not intersegment interactions. Implementing this concept computationally, the domains in a large representative set of proteins were identified; all exhibit consistency with experimental findings. Specifically, our domain divisions correspond to the experimentally determined equilibrium folding intermediates in a set of nine proteins. The approach was also proofed against a representative set of 71 additional proteins, again with confirmatory results. Our reframed interpretation of a protein domain transforms an indeterminate structural phenomenon into a quantifiable molecular property grounded in solution thermodynamics.protein folding | protein structure | protein architecture | protein parsing D omains are visually arresting protein substructures with an influential history in protein biochemistry (1). These familiar, self-contained structural units were first noticed in some of the earliest solved protein structures (2, 3) and soon came to be recognized as common features of protein architecture (4).Dissecting proteins into their constituent domains provides a simple, intuitive approach to classifying protein structure, a molecular application of the time-honored principle of "carving nature at its joints" (5). Many structure-based computer algorithms have been devised to parse the ever-increasing number of solved proteins into discrete units; a highly abbreviated sample includes (6-13). Today, CATH (14) and SCOP (15) are the two most widely used domain classifications. Both are based on computational algorithms but rely ultimately on the human eye as the final arbiter of domain boundaries.However, seeing can be deceiving. The dependence on visual intuition introduces an unavoidable element of ambiguity into procedures for domain recognition. The most enduring domain definition, "potentially independent, stable folding uni...

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“…The TIM unfolding pathways induced by guanidinium hydrochloride (Gdn-HCl) from Bacillus stearothermophilus (25), Thermotoga maritima (26), rabbit (27)(28)(29), Plasmodium falciparum (30), Saccharomyces cereVisiae (31)(32)(33), Leishmania mexicana (34), and Trypanosoma brucei (TbTIM) (35) have been studied in detail. Although the equilibrium unfolding pathways of homologous TIMs in Gdn-HCl are different, the crystallographic threedimensional structures of these enzymes are highly similar.…”

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confidence: 99%

Reversible Equilibrium Unfolding of Triosephosphate Isomerase from Trypanosoma cruzi in Guanidinium Hydrochloride Involves Stable Dimeric and Monomeric Intermediates

et al. 2005

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The reversible guanidinium hydrochloride-induced unfolding of Trypanosoma cruzi triosephosphate isomerase (TcTIM) was characterized under equilibrium conditions. The catalytic activity was followed as a native homodimeric functional probe. Circular dichroism, intrinsic fluorescence, and size-exclusion chromatography were used as secondary, tertiary, and quaternary structural probes, respectively. The change in ANS fluorescence intensity with increasing denaturant concentrations was also determined. The results show that two stable intermediates exist in the transition from the homodimeric native enzyme to the unfolded monomers: one (N(2*)) is a slightly more expanded, non-native, and active dimer, and the other is a partially expanded monomer (M) that binds ANS. Spectroscopic and activity data were used to reach a thermodynamic characterization. The results indicate that the Gibbs free energies for the partial reactions are 4.5 (N(2) <==> N(2*)), 65.8 (N(2*) <==> 2M), and 17.8 kJ/mol (M <==> U). It appears that TcTIM monomers are more stable than those found for other TIM species (except yeast TIM), where monomer stability is only marginal. These results are compared with those for the guanidinium hydrochloride-induced denaturation of TIM from different species, where despite the functional and three-dimensional similarities, a remarkable heterogeneity exists in the unfolding pathways.

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Equilibrium and Kinetic Folding of Rabbit Muscle Triosephosphate Isomerase by Hydrogen Exchange Mass Spectrometry

Cited by 39 publications

References 31 publications

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

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

A thermodynamic definition of protein domains

Reversible Equilibrium Unfolding of Triosephosphate Isomerase from Trypanosoma cruzi in Guanidinium Hydrochloride Involves Stable Dimeric and Monomeric Intermediates

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