Reversible formation of on‐pathway macroscopic aggregates during the folding of maltose binding protein

Ganesh, C.; Zaidi, Faisal N.; Udgaonkar, Jayant B.; Varadarajan, Raghavan

doi:10.1110/ps.8101

Cited by 29 publications

(34 citation statements)

References 36 publications

(37 reference statements)

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“…8 Scheme 1, in addition, incorporates the rate k off [IS]. In Scheme 1, if the binding and dissociation of the intermediate (I) to SecB (S) are assumed to be considerably faster than the transition between I and the native/native-like state N ⁎ , 15,26 preequilibrium will be achieved between I and IS prior to the rate-controlling transition of I to N ⁎ . Based on the preequilibrium assumption and also assuming that refolding in the presence and in the absence of SecB is described by a single exponential, the observed rate constant of folding k app is given by:…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

SecB-Mediated Protein Export Need Not Occur via Kinetic Partitioning

Krishnan

Kulothungan

Patra

et al. 2009

Journal of Molecular Biology

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Section: Resultsmentioning

confidence: 99%

“…1). The activity of refolded MBP was assayed by its ability to bind maltose as described previously, 26 and the refolded protein had activity identical to that of the native protein. k app values for MBP in the presence of SecB were fitted to Eqs.…”

Section: Resultsmentioning

confidence: 99%

SecB-Mediated Protein Export Need Not Occur via Kinetic Partitioning

Krishnan

Kulothungan

Patra

et al. 2009

Journal of Molecular Biology

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“…When unfolded MBP is placed into native conditions, we find that it rapidly adopts a dynamic collapsed state, which can lead to aggregation in vitro when the concentration is >1 μM and to inclusion body formation in vivo (22). Folding to the native state occurs much more slowly even in the absence of aggregation, with all molecules moving through one or more intermediate states to the native state.…”

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confidence: 92%

Folding of a large protein at high structural resolution

Walters

Mayne²,

Hinshaw³

et al. 2013

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

107

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Kinetic folding of the large two-domain maltose binding protein (MBP; 370 residues) was studied at high structural resolution by an advanced hydrogen-exchange pulse-labeling mass-spectrometry method (HX MS). Dilution into folding conditions initiates a fast molecular collapse into a polyglobular conformation (<20 ms), determined by various methods including small angle X-ray scattering. The compaction produces a structurally heterogeneous state with widespread low-level HX protection and spectroscopic signals that match the equilibrium melting posttransition-state baseline. In a much slower step (7-s time constant), all of the MBP molecules, although initially heterogeneously structured, form the same distinct helix plus sheet folding intermediate with the same time constant. The intermediate is composed of segments that are distant in the MBP sequence but adjacent in the native protein where they close the longest residue-to-residue contact. Segments that are most HX protected in the early molecular collapse do not contribute to the initial intermediate, whereas the segments that do participate are among the less protected. The 7-s intermediate persists through the rest of the folding process. It contains the sites of three previously reported destabilizing mutations that greatly slow folding. These results indicate that the intermediate is an obligatory step on the MBP folding pathway. MBP then folds to the native state on a longer time scale (∼100 s), suggestively in more than one step, the first of which forms structure adjacent to the 7-s intermediate. These results add a large protein to the list of proteins known to fold through distinct native-like intermediates in distinct pathways.SAXS | HDX | protein collapse | denatured state ensemble F ifty years after Anfinsen's seminal demonstration that an unfolded protein can refold spontaneously when placed under native conditions, major questions concerning the folding process remain unanswered (1, 2). What is the unfolded state like, its degree of compaction, the reality and character of residual structure before folding begins, and its possible role in guiding the folding process (3-7)? Analogous questions relate to folding intermediates and the folding pathway itself. Do proteins fold through many alternative independent pathways as earlier theoretical investigations have suggested (8-12), or do they fold through necessary intermediates in a distinct pathway (13), as a growing list of experimental observations indicate (14, 15)? To answer these questions, it will be necessary to define experimentally the intermediate forms that proteins move through on their way to the native state. The problem has been that these transient states are beyond the reach of the usual high-resolution crystallographic and NMR structural methods. Most experimental folding studies have therefore relied on low-resolution optical methods that can follow folding in real time but rarely provide the structural information necessary to resolve the basic mechanistic questions.Recent work...

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“…Proteins very often aggregate transiently during folding (29)(30)(31) as well as unfolding (32) because of the self-association of folding or unfolding intermediates, and in some cases the aggregation is specific, leading to the formation of amyloid protofibrils (33,34). In the case of some proteins, amyloid formation is linked directly to disease (35).…”

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Characterization of the Folding and Unfolding Reactions of Single-Chain Monellin: Evidence for Multiple Intermediates and Competing Pathways

2007

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The mechanisms of folding and unfolding of the small plant protein monellin have been delineated in detail. For this study, a single-chain variant of the natively two-chain monellin, MNEI, was used, in which the C terminus of chain B was connected to the N terminus of chain A by a Gly-Phe linker. Equilibrium guanidine hydrochloride (GdnHCl)-induced unfolding experiments failed to detect any partially folded intermediate that is stable enough to be populated at equilibrium to a significant extent. Kinetic experiments in which the refolding of GdnHCl-unfolded protein was monitored by measurement of the change in the intrinsic tryptophan fluorescence of the protein indicated the accumulation of three transient partially structured folding intermediates. The fluorescence change occurred in three kinetic phases: very fast, fast, and slow. It appears that the fast and slow changes in fluorescence occur on competing folding pathways originating from one unfolded form and that the very fast change in fluorescence occurs on a third parallel pathway originating from a second unfolded form of the protein. Kinetic experiments in which the refolding of alkali-unfolded protein was monitored by the change in the fluorescence of the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid (ANS), consequent to the dye binding to the refolding protein, as well as by the change in intrinsic tryptophan fluorescence, not only confirmed the presence of the three kinetic intermediates but also indicated the accumulation of one or more early intermediates at a few milliseconds of refolding. These experiments also exposed a very slow kinetic phase of refolding, which was silent to any change in the intrinsic tryptophan fluorescence of the protein. Hence, the spectroscopic studies indicated that refolding of single-chain monellin occurs in five distinct kinetic phases. Double-jump, interrupted-folding experiments, in which the accumulation of folding intermediates and native protein during the folding process could be determined quantitatively by an unfolding assay, indicated that the fast phase of fluorescence change corresponds to the accumulation of two intermediates of differing stabilities on competing folding pathways. They also indicated that the very slow kinetic phase of refolding, identified by ANS binding, corresponds to the formation of native protein. Kinetic experiments in which the unfolding of native protein in GdnHCl was monitored by the change in intrinsic tryptophan fluorescence indicated that this change occurs in two kinetic phases. Double-jump, interrupted-unfolding experiments, in which the accumulation of unfolding intermediates and native protein during the unfolding process could be determined quantitatively by a refolding assay, indicated that the fast unfolding phase corresponds to the formation of fully unfolded protein via one unfolding pathway and that the slow unfolding phase corresponds to a separate unfolding pathway populated by partially unfolded intermediates. It is shown that the unfolded form produced by...

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Reversible formation of on‐pathway macroscopic aggregates during the folding of maltose binding protein

Cited by 29 publications

References 36 publications

SecB-Mediated Protein Export Need Not Occur via Kinetic Partitioning

SecB-Mediated Protein Export Need Not Occur via Kinetic Partitioning

Folding of a large protein at high structural resolution

Characterization of the Folding and Unfolding Reactions of Single-Chain Monellin: Evidence for Multiple Intermediates and Competing Pathways

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