Helix Mutations Stabilize a Late Productive Intermediate on the Folding Pathway of Ubiquitin

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

doi:10.1021/bi800722d

Cited by 8 publications

(13 citation statements)

References 49 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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“…We also observed a pronounced rollover in the refolding data for the IfN transition which has an initially positive slope for m IN in contrast to the denaturant-independent initial rate profile for k IN observed for FN and WN (m IN e 0) (Figure 7b). This suggested that the intermediate was required to partially unfold to reach the rate-limiting transition state, consistent with a highly compact and misfolded species (27,28). We do not, however, observe the additional refolding and unfolding phases for L8W that are indicative of the more complex kinetics identified for WN.…”

Section: T H I S C O N T E N T Imentioning

confidence: 52%

“…We also considered whether the complex kinetics observed for the WN mutant could be reproduced in the wt-Ub sequence within the context of the native G-bulged type I turn by introducing the L8W mutation in to wt-Ub (TLTGKfTWTGK) (28). This resulted in a highly fluorescent protein that similarly underwent a large change in fluorescence intensity upon folding.…”

Section: T H I S C O N T E N T Imentioning

confidence: 99%

“…These ubiquitin mutants, with enhanced fluorescence of the I-state, resulted in the population of a stable intermediate that was compact, misfolded, and on the folding pathway. Although the various transitions appeared to be ill-resolved for wt-Ub containing the F45W reporter, a series of helix mutants with incremental stabilization of the native state through changes to the structural propensity of the main helix (largely through Ala substitutions at solvent exposed sites) resulted in the I-state also becoming more significantly populated with both the UfI and IfN transitions better-resolved (28). The poor visibility of the I-state in the folding of wt-Ub F45W mutant is most likely associated with the fact that it is only weakly populated.…”

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confidence: 99%

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Aromatic Residues Engineered into the β-Turn Nucleation Site of Ubiquitin Lead to a Complex Folding Landscape, Non-Native Side-Chain Interactions, and Kinetic Traps

et al. 2008

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The fast folding of small proteins is likely to be the product of evolutionary pressures that balance the search for native-like contacts in the transition state with the minimum number of stable non-native interactions that could lead to partially folded states prone to aggregation and amyloid formation. We have investigated the effects of non-native interactions on the folding landscape of yeast ubiquitin by introducing aromatic substitutions into the beta-turn region of the N-terminal beta-hairpin, using both the native G-bulged type I turn sequence (TXTGK) as well as an engineered 2:2 XNGK type I' turn sequence. The N-terminal beta-hairpin is a recognized folding nucleation site in ubiquitin. The folding kinetics for wt-Ub (TLTGK) and the type I' turn mutant (TNGK) reveal only a weakly populated intermediate, however, substitution with X = Phe or Trp in either context results in a high propensity to form a stable compact intermediate where the initial U-->I collapse is visible as a distinct kinetic phase. The introduction of Trp into either of the two host turn sequences results in either complex multiphase kinetics with the possibility of parallel folding pathways, or formation of a highly compact I-state stabilized by non-native interactions that must unfold before refolding. Sequence substitutions with aromatic residues within a localized beta-turn capable of forming non-native hydrophobic contacts in both the native state and partially folded states has the undesirable consequence that folding is frustrated by the formation of stable compact intermediates that evolutionary pressures at the sequence level may have largely eliminated.

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“…Here, we investigate the thermodynamics and kinetics of Ub folding, which has been investigated by a variety of experiments − and computations. ,− However, the effects of denaturants, such as guanidinium chloride and urea, have not been considered in simulations. This is important because there are few central experimental controversies, such as the extent of collapse in the denatured ensemble of Ub as the denaturant concentration is lowered and if intermediates are present, that remain unresolved.…”

Section: Introductionmentioning

confidence: 99%

Collapse Precedes Folding in Denaturant-Dependent Assembly of Ubiquitin

Reddy

Thirumalai

2017

J. Phys. Chem. B

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The folding of small protein ubiquitin (Ub), which plays an indispensable role in targeting proteins for degradation and DNA damage response, is complex. A number of experiments on Ub folding have reached differing conclusions regarding the relation between collapse and folding, and whether intermediates are populated. In order to resolve these vexing issues, we elucidate the denaturant-dependent thermodynamics and kinetics of Ub folding at low and neutral pH as a function of guanidinium chloride and urea using coarse-grained molecular simulations. The changes in the fraction of the folded Ub, and the radius of gyration (R) as a function of the denaturant concentration, [C], are in quantitative agreement with experiments. Under conditions used in experiments, R of the unfolded state at neutral pH changes only by ≈17% as the [GdmCl] decreases from 6 to 0 M. We predict that the extent of compaction of the unfolded state increases as temperature decreases. A two-dimensional folding landscape as a function of R and a measure of similarity to the folded state reveals unambiguously that the native state assembly is preceded by collapse, as discovered in fast mixing experiments on several proteins. Analyses of the folding trajectories, under mildly denaturing conditions ([GdmCl] = 1.0 M or [Urea] = 1.0 M), shows that Ub folds by collision between preformed secondary structural elements involving kinetic intermediates that are primarily stabilized by long-range contacts. Our work explains the results of small angle X-ray scattering (SAXS) experiments on Ub quantitatively, and establishes that evolved globular proteins in the unfolded ensemble are poised to collapse as the solvent conditions for the biopolymer changes from good solvent to Θ-solvent like conditions on denaturant dilution. In the process, we explain the discrepancy between SAXS and single molecule fluorescent resonant energy transfer (smFRET) experiments, which have arrived at a contradicting conclusion concerning the collapse of polypeptide chains.

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Helix Mutations Stabilize a Late Productive Intermediate on the Folding Pathway of Ubiquitin

Cited by 8 publications

References 49 publications

Plant Coilin: Structural Characteristics and RNA-Binding Properties

Plant Coilin: Structural Characteristics and RNA-Binding Properties

Aromatic Residues Engineered into the β-Turn Nucleation Site of Ubiquitin Lead to a Complex Folding Landscape, Non-Native Side-Chain Interactions, and Kinetic Traps

Collapse Precedes Folding in Denaturant-Dependent Assembly of Ubiquitin

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