Structural basis for unfolding pathway‐dependent stability of proteins: Vectorial unfolding versus global unfolding

Yagawa, Keisuke; Yamano, Koji; Oguro, Takaomi; Sato, Takehiro; Momose, Takumaro; Kawano, Shin; Endo, Toshiya

doi:10.1002/pro.346

Cited by 20 publications

(31 citation statements)

References 23 publications

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“…This result may be due to the fact that the Y79WW83F intermediate resembles an equilibrium intermediate observed for CopC‐Cu(II). In addition, recent research about I27 suggests that the mutated form of I27 causes a bend in the backbone structure, which leads to the formation of a more stable N‐terminal structure probably through enhanced hydrophobic interactions . The unfolding curve of the Y79W induced by urea also shows two transitions.…”

Section: Discussionmentioning

confidence: 97%

“…In addition, recent research about I27 suggests that the mutated form of I27 causes a bend in the backbone structure, which leads to the formation of a more stable N-terminal structure probably through enhanced hydrophobic interactions. 40 The unfolding curve of the Y79W induced by urea also shows two transitions. The intermediate ascribed to the mutant was also observed for the pseudo-azurin structure, another protein with the same b-barrel fold as CopC.…”

Section: Discussionmentioning

confidence: 98%

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Conformational stability of CopC and roles of residues Tyr79 and Trp83

2013

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CopC is a periplasmic copper Chaperone protein that has a b-barrel fold and two metalbinding sites distinct for Cu(II) and Cu(I). In the article, four mutants (Y79F, Y79W, Y79WW83L, Y79WW83F) were obtained by site-directed mutagenesis. The far-UV CD spectra of the proteins were similar, suggesting that mutations did not bring any significant changes in secondary structures. Meanwhile the effects of mutations on the protein's function were manifested by Cu(II) binding. Fluorescence lifetime measurement and quenching of tryptophan fluorescence by acrylamide and KI showed that the microenvironment around Trp83 was more hydrophobic than that around Tyr79 in apoCopC. Unfolding experiments induced by guanidinium chloride (GdnHCl), urea provided the conformational stability of each protein. The D obtained using the model of structural elements was used to show the role of Tyr79 and Trp83. On the one hand, the induced by urea for Y79F, Y79W have a loss of 6.51, 2.03 kJ=mol, respectively, compared with apoCopC, proving that replacement of Tyr79 by Phe or Trp all decreased the protein stability, meaning that the hydrogen bonds interactions between Tyr79 and Thr75 played an important role in stabilizing apoCopC. On the other hand, the induced by urea for Y79WW83L have a loss of 11.44 kJ=mol, but for Y79WW83F did a raise of 1.82 kJ=mol compared with Y79W. The replacement of Trp83 by Phe and Leu yields opposite effects on protein stability, which suggested that the aromatic ring of Trp83 was important in maintaining the hydrophobic core of apoCopC.

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Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 98%

Conformational stability of CopC and roles of residues Tyr79 and Trp83

2013

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“…Using a combination of MD simulation, force-profile measurements and cryo-EM, we have investigated the cotranslational folding pathway of the 89-residue titin I27 domain. I27 has been extensively characterised in previous in vitro folding studies (40, 41, 45-55). Results from all three techniques show that wild-type I27 folds in the mouth of the ribosome exit tunnel; in the cryo-EM structure of I27-TnaC[ L =35] RNCs, I27 packs against ribosomal proteins uL24, uL29, and ribosomal 23S RNA.…”

Section: Discussionmentioning

confidence: 99%

The Folding Pathway of an Ig Domain is Conserved On and Off the Ribosome

Tian¹,

Steward

Kudva

et al. 2018

Preprint

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Proteins that fold cotranslationally may do so in a restricted configurational space, due to the volume occupied by the ribosome. How does this environment, coupled with the close proximity of the ribosome, affect the folding pathway of a protein? Previous studies have shown that the cotranslational folding process for many proteins, including small, single domains, is directly affected by the ribosome. Here, we investigate the cotranslational folding of an all-b immunoglobulin domain, titin I27. Using an arrest peptide-based assay and structural studies by cryo-EM, we show that I27 folds in the mouth of the ribosome exit tunnel. Simulations that use a kinetic model for the force-dependence of escape from arrest, accurately predict the fraction of folded protein as a function of length. We used these simulations to probe the folding pathway on and off the ribosome. Our simulations - which also reproduce experiments on mutant forms of I27 - show that I27 folds, while still sequestered in the mouth of the ribosome exit tunnel, by essentially the same pathway as free I27, with only subtle shifts of critical contacts from the C to the N terminus.Significance StatementMost proteins need to fold into a specific three-dimensional structure in order to function. The mechanism by which isolated proteins fold has been thoroughly studied by experiment and theory. However, in the cell proteins do not fold in isolation, but are synthesized as linear chains by the ribosome during translation. It is therefore natural to ask at which point during synthesis proteins fold, and whether this differs from the folding of isolated protein molecules. By studying folding of a well characterized protein domain, titin I27, stalled at different points during translation, we show that it already folds in the mouth of the ribosome exit tunnel, and that the mechanism is almost identical to that of the isolated protein.

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“…Multiple papers describe the mechanical unfolding of titin domains [among others: (Rief et al, 1998, 2000; Li and Fernandez, 2003; Garcia et al, 2009; Stacklies et al, 2009; Yagawa et al, 2010)]. FnIII-like structures unfold at lower forces than Ig domains (100–200 pN vs. 150–300 pN) (Rief et al, 1998).…”

Section: Ig and Fniii Domainsmentioning

confidence: 99%

Structure of giant muscle proteins

Meyer

Wright

2013

Front. Physiol.

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Giant muscle proteins (e.g., titin, nebulin, and obscurin) play a seminal role in muscle elasticity, stretch response, and sarcomeric organization. Each giant protein consists of multiple tandem structural domains, usually arranged in a modular fashion spanning 500 kDa to 4 MDa. Although many of the domains are similar in structure, subtle differences create a unique function of each domain. Recent high and low resolution structural and dynamic studies now suggest more nuanced overall protein structures than previously realized. These findings show that atomic structure, interactions between tandem domains, and intrasarcomeric environment all influence the shape, motion, and therefore function of giant proteins. In this article we will review the current understanding of titin, obscurin, and nebulin structure, from the atomic level through the molecular level.

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Structural basis for unfolding pathway‐dependent stability of proteins: Vectorial unfolding versus global unfolding

Cited by 20 publications

References 23 publications

Conformational stability of CopC and roles of residues Tyr79 and Trp83

Conformational stability of CopC and roles of residues Tyr79 and Trp83

The Folding Pathway of an Ig Domain is Conserved On and Off the Ribosome

Structure of giant muscle proteins

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