Population of On-pathway Intermediates in the Folding of Ubiquitin

Crespo, Maria D.; Simpson, Emma R.; Searle, Mark S.

doi:10.1016/j.jmb.2006.05.061

Cited by 24 publications

(43 citation statements)

References 73 publications

(131 reference statements)

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“…Thus, the fast phase is well resolved from the slower phases and the kinetic traces can be fit to multi-exponential functions to obtain accurate rate constants for the major folding phase. It should be noted that the Searle group has recently shown that the slow phases observed for the folding of an engineered variant of yeast ubiquitin may be attributed to the formation of an on-pathway intermediate [39], at the present time, there is no evidence for this intermediate with mammalian ubiquitin ( [35][36][37][38] and the two-state model is therefore used here.…”

Section: General Conditionsmentioning

confidence: 99%

“…Positive cross-interaction parameters indicate the absence of parallel pathways for folding [55]. Although parallel folding pathways have been suggested for yeast sequence ubiquitin [39], studies in the Jackson laboratory have not suggested parallel pathways exist for mammalian ubiquitin, and Ψ-value transition state analysis suggests a single pathway is traversed in the transition-state ensemble [56]. The ratelimiting step for refolding of two-state proteins is collapse of the denatured state [38], and a change in rate-limiting step would manifest itself as premature collapse of the coil as observed with CI2 [29].…”

Section: Movement Of the Denatured State Along The Reaction Coordinatementioning

confidence: 99%

See 1 more Smart Citation

Destabilised mutants of ubiquitin gain equal stability in crowded solutions

Roberts¹,

Jackson²

2007

Biophysical Chemistry

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT AbstractThis paper investigates the thermodynamic and kinetic response of WT* ubiquitin (F45W) and three mutants to high concentrations of glucose, sucrose and dextran under physiological temperature and pH. WT* ubiquitin was stabilised by the same amount when comparing each cosolute on a weight to volume ratio, with cosolute effects largely independent of denaturant concentration. The energy difference between the mutants and WT* ubiquitin also remained the same in high concentrations of cosolute. An apparent decrease in transition-state surface burial in the presence of the cosolutes was attributed to increased compaction of the denatured state, and not to the Hammond effect. Together, these results suggest higher thermodynamic stabilities and folding rates for proteins in vivo compared to in vitro, in addition to more compact denatured states. Because the effects of mutation are the same in dilute solution and crowded conditions used to mimic the cellular environment, there is validity in using measurements of mutant stabilities made in dilute solutions to inform on how the mutations may affect stability in vivo.

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Section: General Conditionsmentioning

confidence: 99%

Section: Movement Of the Denatured State Along The Reaction Coordinatementioning

confidence: 99%

Destabilised mutants of ubiquitin gain equal stability in crowded solutions

Roberts¹,

Jackson²

2007

Biophysical Chemistry

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“…Observing the multitude of trajectories and folding structures using bulk techniques averages out the ensembles predicted by the statistical theories of protein folding. Much of the literature studying bulk denaturation and refolding of proteins therefore reports 2-state folding reactions where even the presence of single folding intermediates is controversial (18,19). Indeed, as pointed out by Dill and Chan (15) in their seminal review, the predictions made by the statistical theories are in conflict with the view that folding proceeds through a well defined pathway that crosses a single energy barrier to the native state.…”

mentioning

confidence: 99%

Direct observation of an ensemble of stable collapsed states in the mechanical folding of ubiquitin

Garcia-Manyes

Dougan

Badilla

et al. 2009

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

115

163

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Statistical theories of protein folding have long predicted plausible mechanisms for reducing the vast conformational space through distinct ensembles of structures. However, these predictions have remained untested by bulk techniques, because the conformational diversity of folding molecules has been experimentally unapproachable. Owing to recent advances in single molecule force-clamp spectroscopy, we are now able to probe the structure and dynamics of the small protein ubiquitin by measuring its length and mechanical stability during each stage of folding. Here, we discover that upon hydrophobic collapse, the protein rapidly selects a subset of minimum energy structures that are mechanically weak and essential precursors of the native fold. From this much reduced ensemble, the native state is acquired through a barrier-limited transition. Our results support the validity of statistical mechanics models in describing the folding of a small protein on biological timescales.force-clamp spectroscopy ͉ protein folding M ore than 2 decades ago statistical theories of protein folding, developed from simplified models of proteins (1) and scaling laws borrowed from polymer physics (2), offered an appealing framework for how proteins fold (3-5). These pioneering studies laid down the foundation of the ''new view'' of the protein folding theory (6-8). From this viewpoint, a polypeptide exposed to folding conditions is thought to go through progressively smaller conformational ensembles along a rough, funnel-like energy surface leading to the native state topology (8, 9). The envisaged stages of folding involve an initial hydrophobic collapse into a large set of nonspecific globular structures, which then select a few minimum energy collapsed conformations that fold via activated all-or-none transitions (4,5,(10)(11)(12)(13)(14). Time scales for each one of these phases were estimated from simplified models to range from microseconds for the collapse to seconds for the final fold (2,12). Although theoretical models propose physical mechanisms underlying protein folding (12,(15)(16)(17), their experimental verification has been inaccessible. Observing the multitude of trajectories and folding structures using bulk techniques averages out the ensembles predicted by the statistical theories of protein folding. Much of the literature studying bulk denaturation and refolding of proteins therefore reports 2-state folding reactions where even the presence of single folding intermediates is controversial (18,19). Indeed, as pointed out by Dill and Chan (15) in their seminal review, the predictions made by the statistical theories are in conflict with the view that folding proceeds through a well defined pathway that crosses a single energy barrier to the native state. This longstanding debate can only be addressed by sampling the conformational diversity of a single protein along its folding trajectory.Here, we demonstrate that single protein force-clamp spectroscopy using the AFM captures the diversity of the collapsed conforma...

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“…All experiments were performed in 25 mM sodium acetate buffer (pH 5.0) and five traces were averaged for each experiment. 10,54,57 The kinetic data were analysed using multiexponential fitting procedures (two refolding phases and one unfolding phase were observed) with the quality of the fit determined from an analysis of the residuals. 10 A slow refolding phase was observed over a wide range of conditions that showed a very low amplitude (b5%) and little variation with denaturant concentration, suggesting a slow isomerisation-limited phase.…”

Section: Kinetic Experimentsmentioning

confidence: 99%