Multiple Tryptophan Probes Reveal that Ubiquitin Folds via a Late Misfolded Intermediate

Vallée‐Bélisle, Alexis; Michnick, Stephen W.

doi:10.1016/j.jmb.2007.09.018

Cited by 31 publications

(43 citation statements)

References 69 publications

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“…These data demonstrate that this α-helix might stabilize the NOD tertiary structure. The result supports the model suggested above, since the α-helix was shown to be the crucial element in the formation of the ubiquitin-fold [41], [42].…”

Section: Resultssupporting

confidence: 90%

Plant Coilin: Structural Characteristics and RNA-Binding Properties

et al. 2013

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Cajal bodies (CBs) are dynamic subnuclear compartments involved in the biogenesis of ribonucleoproteins. Coilin is a major structural scaffolding protein necessary for CB formation, composition and activity. The predicted secondary structure of Arabidopsis thaliana coilin (Atcoilin) suggests that the protein is composed of three main domains. Analysis of the physical properties of deletion mutants indicates that Atcoilin might consist of an N-terminal globular domain, a central highly disordered domain and a C-terminal domain containing a presumable Tudor-like structure adjacent to a disordered C terminus. Despite the low homology in amino acid sequences, a similar type of domain organization is likely shared by human and animal coilin proteins and coilin-like proteins of various plant species. Atcoilin is able to bind RNA effectively and in a non-specific manner. This activity is provided by three RNA-binding sites: two sets of basic amino acids in the N-terminal domain and one set in the central domain. Interaction with RNA induces the multimerization of the Atcoilin molecule, a consequence of the structural alterations in the N-terminal domain. The interaction with RNA and subsequent multimerization may facilitate coilin’s function as a scaffolding protein. A model of the N-terminal domain is also proposed.

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Section: Resultssupporting

confidence: 90%

Plant Coilin: Structural Characteristics and RNA-Binding Properties

et al. 2013

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“…Our kinetic studies show that both wt-ub and (4R)-FPro-ub fold following a sequential three-state model mechanism that involves the transient population of an obligatory on-pathway intermediate, and are fully in agreement with previously reported studies on ubiquitin folding [30], [35], [36]. The presence of intermediates in the folding mechanism of ubiquitin has been previously reported in numerous studies [27], [30], [35], [37] and in our study we observed that the intermediate for both wt-ub and (4R)-FPro-ub was populated below 1% at low denaturant concentrations. A global fit of the kinetic and equilibrium data to an on-pathway three-state model is shown in Figure 4b and data extracted from best fit is summarized in Table 2.…”

Section: Resultssupporting

confidence: 92%

Rational Design of Protein Stability: Effect of (2S,4R)-4-Fluoroproline on the Stability and Folding Pathway of Ubiquitin

Crespo

Rubini

2011

PLoS ONE

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BackgroundMany strategies have been employed to increase the conformational stability of proteins. The use of 4-substituted proline analogs capable to induce pre-organization in target proteins is an attractive tool to deliver an additional conformational stability without perturbing the overall protein structure. Both, peptides and proteins containing 4-fluorinated proline derivatives can be stabilized by forcing the pyrrolidine ring in its favored puckering conformation. The fluorinated pyrrolidine rings of proline can preferably stabilize either a Cγ-exo or a Cγ-endo ring pucker in dependence of proline chirality (4R/4S) in a complex protein structure. To examine whether this rational strategy can be generally used for protein stabilization, we have chosen human ubiquitin as a model protein which contains three proline residues displaying Cγ-exo puckering.Methodology/Principal FindingsWhile (2S,4R)-4-fluoroproline ((4R)-FPro) containing ubiquitinin can be expressed in related auxotrophic Escherichia coli strain, all attempts to incorporate (2S,4S)-4-fluoroproline ((4S)-FPro) failed. Our results indicate that (4R)-FPro is favoring the Cγ-exo conformation present in the wild type structure and stabilizes the protein structure due to a pre-organization effect. This was confirmed by thermal and guanidinium chloride-induced denaturation profile analyses, where we observed an increase in stability of −4.71 kJ·mol−1 in the case of (4R)-FPro containing ubiquitin ((4R)-FPro-ub) compared to wild type ubiquitin (wt-ub). Expectedly, activity assays revealed that (4R)-FPro-ub retained the full biological activity compared to wt-ub.Conclusions/SignificanceThe results fully confirm the general applicability of incorporating fluoroproline derivatives for improving protein stability. In general, a rational design strategy that enforces the natural occurring proline puckering conformation can be used to stabilize the desired target protein.

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“…Major strategies range from characterizing transient intermediates during refolding experiments to studying equilibrium species populated through the use of unnatural conditions (e.g., low pH, cosolvents) or mutagenesis [96–99]. Another major strategy involves the detection of sparsely populated equilibrium intermediates, for example, using NSHX and NMR RD methods [78–81,100–102].…”

Section: Intermediates and Desolvationmentioning

confidence: 99%

The folding of single domain proteins—have we reached a consensus?

Sosnick

Barrick

2011

Current Opinion in Structural Biology

135

130

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Rather than stressing the most recent advances in the field, this review highlights the fundamental topics where disagreement remains and where adequate experimental data are lacking. These topics include properties of the denatured state and the role of residual structure, the nature of the fundamental steps and barriers, the extent of pathway heterogeneity and non-native interactions, recent comparisons between theory and experiment, and finally, dynamical properties of the folding reaction.

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Multiple Tryptophan Probes Reveal that Ubiquitin Folds via a Late Misfolded Intermediate

Cited by 31 publications

References 69 publications

Plant Coilin: Structural Characteristics and RNA-Binding Properties

Plant Coilin: Structural Characteristics and RNA-Binding Properties

Rational Design of Protein Stability: Effect of (2S,4R)-4-Fluoroproline on the Stability and Folding Pathway of Ubiquitin

The folding of single domain proteins—have we reached a consensus?

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