The Folding Pathway of Barnase:  The Rate-Limiting Transition State and a Hidden Intermediate under Native Conditions

Vu, Ngoc-Diep; Feng, Hanqiao; Bai, Yawen

doi:10.1021/bi0362267

Cited by 50 publications

(54 citation statements)

References 52 publications

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“…This partially structured intermediate is unstable and would not be expected to accumulate during the folding reaction and, in this respect, is different from those folding intermediates that are traditionally defined and identified by their accumulated populations (17,18). Instead, the intermediates observed in the present study are more akin to the sparsely populated ''hidden'' intermediates inferred from native-state hydrogen-exchange experiments (4,19,20). Similarly, a partially unstructured intermediate on the protein's unfolding pathway was identified in trajectories of the folded protein.…”

contrasting

confidence: 52%

Sparsely populated folding intermediates of the Fyn SH3 domain: Matching native-centric essential dynamics and experiment

Ollerenshaw

Kaya

Chan

et al. 2004

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

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contrasting

confidence: 52%

Sparsely populated folding intermediates of the Fyn SH3 domain: Matching native-centric essential dynamics and experiment

Ollerenshaw

Kaya

Chan

et al. 2004

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

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“…Thermodynamic principle requires that proteins repeatedly unfold and refold even under native conditions, revisiting their normal folding intermediates and recapitulating their normal folding process. All or parts of this process have been observed by site-resolved hydrogen exchange (19,(29)(30)(31)(32)(33)(34)(35), by a related thiol reactivity method (36), by NMR relaxation dispersion (37)(38)(39), and by theoretical analysis (40). Stable on-pathway intermediates built from cooperative foldon elements of the native protein are seen even under two-state folding conditions.…”

Section: Discussionmentioning

confidence: 99%

Protein folding: Independent unrelated pathways or predetermined pathway with optional errors

Bédard

Mallela

Mayne

et al. 2008

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

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The observation of heterogeneous protein folding kinetics has been widely interpreted in terms of multiple independent unrelated pathways (IUP model), both experimentally and in theoretical calculations. However, direct structural information on folding intermediates and their properties now indicates that all of a protein population folds through essentially the same stepwise pathway, determined by cooperative native-like foldon units and the way that the foldons fit together in the native protein. It is essential to decide between these fundamentally different folding mechanisms. This article shows, contrary to previous supposition, that the heterogeneous folding kinetics observed for the staphylococcal nuclease protein (SNase) does not require alternative parallel pathways. SNase folding kinetics can be fit equally well by a single predetermined pathway that allows for optional misfolding errors, which are known to occur ubiquitously in protein folding. Structural, kinetic, and thermodynamic information for the folding intermediates and pathways of many proteins is consistent with the predetermined pathway-optional error (PPOE) model but contrary to the properties implied in IUP models.foldons ͉ PPOE model ͉ staphylococcal nuclease T hat proteins might fold by way of some predetermined pathway was first suggested by C. Levinthal (1), who reasoned that folding through a random search process would take far longer than the subsecond time scale often observed. Experimental work driven by this concept found evidence for distinct pathway intermediates (2). However, theoretical work showed that the energetically downhill nature of the conformational search might by itself be sufficient to drive random folding on a realistic time scale (3). No specific pathway would then be necessary. This scenario has been formalized in the funneled energy landscape view for protein folding (4-8). Following this precedent, experimental results for a number of proteins have been similarly interpreted in terms of multiple unrelated parallel pathways (9)(10)(11)(12)(13)(14)(15)(16). The critical observation is that protein folding can be kinetically heterogeneous. Different fractions of a folding population fold at different rates and exhibit different populated intermediates, suggesting alternative pathways. This view is often represented in terms of multiple tracks through the funneled energy landscape. We refer to this view as the independent unrelated pathways (IUP) model to emphasize that intermediate structures in the different tracks have no particular relationship.A much more determinate pathway is supported by structural information derived experimentally for folding intermediates in many proteins (17)(18)(19)(20). This information indicates that protein folding intermediates are definitively native-like, constructed from cooperative foldon units of the target native protein. It appears that native-like foldon units are formed and docked into place one after another in a stepwise sequence, each one guided and stabilized by pr...

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“…Also, local unfolding events that result in complete collapse of the protein are generally not observed in solution-based protein unfolding studies. Native state hydrogen exchange studies on barnase (the substrate used in mitochondrial import studies) (27), show that local structure may unfold as a cooperative unit, but these local-unfolding or fraying events are not rate-limiting to unfolding in solution. Although the mechanical unfolding energy landscape may differ from that encountered in solution, it seems unlikely that the unfolding of a single ␣ helix or loop on the surface of a protein represents its rate-limiting structure or mechanical breakpoint.…”

Section: Does Local Structure Control Translocation-coupled Unfolding?mentioning

confidence: 99%

Lethal factor unfolding is the most force-dependent step of anthrax toxin translocation

Thoren¹,

Worden

Yassif

et al. 2009

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

125

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Cellular compartmentalization requires machinery capable of translocating polypeptides across membranes. In many cases, transported proteins must first be unfolded by means of the proton motive force and/or ATP hydrolysis. Anthrax toxin, which is composed of a channel-forming protein and two substrate proteins, is an attractive model system to study translocation-coupled unfolding, because the applied driving force can be externally controlled and translocation can be monitored directly by using electrophysiology. By controlling the driving force and introducing destabilizing point mutations in the substrate, we identified the barriers in the transport pathway, determined which barrier corresponds to protein unfolding, and mapped how the substrate protein unfolds during translocation. In contrast to previous studies, we find that the protein's structure next to the signal tag is not rate-limiting to unfolding. Instead, a more extensive part of the structure, the amino-terminal ␤-sheet subdomain, must disassemble to cross the unfolding barrier. We also find that unfolding is catalyzed by the channel's phenylalanine-clamp active site. We propose a broad molecular mechanism for translocation-coupled unfolding, which is applicable to both soluble and membrane-embedded unfolding machines.unfolding pathway ͉ transition state structure ͉ mechanical unfolding F olded proteins are Ϸ5-10 kcal⅐mol Ϫ1 more stable than their unfolded states. Therefore, the disassembly and translocation of folded proteins often require a molecular machine and a source of free energy. These ubiquitous multiprotein complexes include soluble degradation machinery, such as the proteasome or the Clp bacterial proteases (1), which unfold and degrade proteins, and some, but not all, membrane-embedded translocase channels, which can unfold and transport proteins across membranes (2). There are general features shared between these soluble and membrane-embedded translocase machines: a narrow central pore first engages the protein substrate on its free end, the substrate is unfolded mechanically, and the unfolded chain is translocated through the narrow pore, allowing it ultimately to either cross a membrane or enter into a proteolytic complex for degradation. Protein unfolding and translocation in these systems are often driven by ATP hydrolysis (1, 2), a membrane potential (⌬⌿) (2, 3), and/or a proton gradient (⌬pH) (4). The molecular mechanism of translocation-coupled unfolding, however, is poorly understood.Prior studies examining the correlation between substrate protein stability and translocation kinetics have produced conflicting results. Some ligand-stabilized substrates translocate inefficiently, because they are too thermodynamically stable (5); however, other substrates show little change in the rate of translocation when destabilized by mutagenesis (6, 7). To resolve these conflicting results, it was proposed that translocation-coupled unfolding (6, 8) depends on the mechanical stability of the local structure adjacent to the signal tag. O...

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The Folding Pathway of Barnase: The Rate-Limiting Transition State and a Hidden Intermediate under Native Conditions

Cited by 50 publications

References 52 publications

Sparsely populated folding intermediates of the Fyn SH3 domain: Matching native-centric essential dynamics and experiment

Sparsely populated folding intermediates of the Fyn SH3 domain: Matching native-centric essential dynamics and experiment

Protein folding: Independent unrelated pathways or predetermined pathway with optional errors

Lethal factor unfolding is the most force-dependent step of anthrax toxin translocation

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