Effect of the C-terminal Truncation on the Functional Cycle of Chaperonin GroEL

Suzuki, Mihoko; Ueno, Taro; Iizuka, Ryo; Miura, Takahiro; Zako, Tamotsu; Akahori, Rena; Miyake, Takeo; Shimamoto, Norio; Aoki, Michiko; Takenaka, Takashi; Ohdomari, Iwao; Funatsu, Takashi

doi:10.1074/jbc.m804090200

Cited by 30 publications

(13 citation statements)

References 35 publications

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“…In this case, the rate of ATP hydrolysis into ADP-Pi in the presence of DM-MBP is 1/(1/k FB-I þ 1/k BF-I þ 1/k 1 ) ¼ 0.32 s 21 or 1/(1/k FB-I þ 1/k BF-I þ 1/k 2 ) ¼ 0.28 s 21 . These values are close to the values estimated from biochemical studies in the presence of foldable SP, 0.3120.36 s 21 [35,43]. Note that the biochemically determined rate of ATP !…”

Section: Discussionsupporting

confidence: 85%

Substrate protein dependence of GroEL–GroES interaction cycle revealed by high-speed atomic force microscopy imaging

Noshiro

Ando

2018

Phil. Trans. R. Soc. B

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A double-ring-shaped tetradecameric GroEL complex assists proper protein folding in cooperation with the cochaperonin GroES. The dynamic GroEL-GroES interaction reflects the allosteric intra- and inter-ring communications and the chaperonin reaction. Therefore, revealing this dynamic interaction is essential to understanding the allosteric communications and the operation mechanism of GroEL. Nevertheless, how this interaction proceeds in the chaperonin cycle has long been controversial. Here, we directly image the dynamic GroEL-GroES interaction under conditions with and without foldable substrate protein using high-speed atomic force microscopy. Then, the imaging results obtained under these conditions and our previous results in the presence of unfoldable substrate are compared. The molecular movies reveal that the entire reaction pathway is highly complicated but basically identical irrespective of the substrate condition. A prominent (but moderate) difference is in the population distribution of intermediate species: symmetric GroEL : GroES and asymmetric GroEL : GroES complexes, and GroES-unbound GroEL. This difference is mainly attributed to the longer lifetime of GroEL : GroES complexes in the presence of foldable substrate. Moreover, the inter-ring communication, which is the basis for the alternating action of the two rings, occurs at two distinct (GroES association and dissociation) steps in the main reaction pathway, irrespective of the substrate condition.This article is part of a discussion meeting issue 'Allostery and molecular machines'.

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Section: Discussionsupporting

confidence: 85%

Substrate protein dependence of GroEL–GroES interaction cycle revealed by high-speed atomic force microscopy imaging

Noshiro

Ando

2018

Phil. Trans. R. Soc. B

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“…A cysteine-introduced GroES variant (ES98C) (17) modified with Alexa Fluor 488 and biotin (termed A488bio-ES) was immobilized in ZMWs through a biotin-streptavidin linkage. We have previously confirmed that the immobilization of GroES does not perturb the binding to GroEL (21). Wild-type GroEL was fluorescently labeled with Cy5 (termed Cy5-EL).…”

Section: Resultsmentioning

confidence: 88%

“…A flow cell was constructed from a glass slide and coverslip with ZMWs separated by two spaces of ϳ50-m thickness (Lumirror 50-S10; Toray Industries, Tokyo, Japan). A488bio-ES was immobilized on to the bottom of the ZMWs through biotinylated bovine serum albumin and streptavidin as previously described (21). Experimental results shown in this paper were obtained in HKM buffer containing 50 nM Cy5-EL, 300 nM Cy3-labeled t-ES 98C (Cy3-ES), 2 mM ATP, 10 M reduced LA (rLA), an ATP-regeneration system (5 mM phosphoenolpyruvate and 10 g/ml pyruvate kinase), an oxygen scavenging system (0.45% (w/v) D-glucose, 50 units/ml glucose oxidase, and 50 units/ml catalase), 7 mM dithiothreitol, and 2 mM Trolox.…”

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confidence: 99%

Single-molecule Study on the Decay Process of the Football-shaped GroEL-GroES Complex Using Zero-mode Waveguides

Sameshima

Iizuka

Ueno

et al. 2010

Journal of Biological Chemistry

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It has been widely believed that an asymmetric GroEL-GroES complex (termed the bullet-shaped complex) is formed solely throughout the chaperonin reaction cycle, whereas we have recently revealed that a symmetric GroEL-(GroES) 2 complex (the football-shaped complex) can form in the presence of denatured proteins. However, the dynamics of the GroEL-GroES interaction, including the football-shaped complex, is unclear. We investigated the decay process of the football-shaped complex at a single-molecule level. Because submicromolar concentrations of fluorescent GroES are required in solution to form saturated amounts of the football-shaped complex, single-molecule fluorescence imaging was carried out using zero-mode waveguides. The single-molecule study revealed two insights into the GroEL-GroES reaction. First, the first GroES to interact with GroEL does not always dissociate from the football-shaped complex prior to the dissociation of a second GroES. Second, there are two cycles, the "football cycle " and the "bullet cycle," in the chaperonin reaction, and the lifetimes of the footballshaped and the bullet-shaped complexes were determined to be 3-5 s and about 6 s, respectively. These findings shed new light on the molecular mechanism of protein folding mediated by the GroEL-GroES chaperonin system.The chaperonin is an essential molecular chaperone that assists protein folding in the cell (1, 2). The Escherichia coli chaperonin GroEL is composed of 14 identical 57-kDa subunits arranged in two heptameric rings stacked back-to-back with each containing a cavity. The cofactor, GroES, consists of a dome-shaped heptameric ring of identical 10-kDa subunits.The widely accepted model of protein folding mediated by the GroEL-GroES chaperonin system is as follows. (i) The denatured protein is injected into one of GroEL rings (cis-ring) upon the ATP-dependent formation of the GroEL-GroES complex.(ii) ATP hydrolysis in the cis-ring results in the formation of the GroEL-GroES complex with bound ADP. (iii) Subsequent ATP binding to the opposite ring (trans-ring) induces the release of GroES, ADP, and the encapsulated protein from the cis-ring.(iv) The trans-ring is reoriented to a new cis-ring, thereby allowing the next ATPase cycle (1-5). In other words, an asymmetric GroEL-GroES complex (termed the bullet-shaped complex) is formed throughout the cycle, whereas a symmetric GroEL-(GroES) 2 complex (termed the football-shaped complex) does not exist.The accepted model has been challenged by the findings that indicate the existence of the football-shaped complex (6 -14). In particular, we have recently revealed that the bullet-and the football-shaped complexes coexist during the reaction cycle (14). We have also found that the formation of the footballshaped complex is regulated by the ATP/ADP ratio (14) and the amount of denatured proteins in solution (13). However, the dynamics of the GroEL-GroES interaction via the footballshaped complex remains elusive. Here, we have developed a single-molecule assay using zero-mode waveguid...

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“…S1A). Previous studies show that in forming a complex with GroEL, GroES inhibits the ATP hydrolysis rate of GroEL by ϳ50% (16,21,45,46). Neither GroESI25D nor GroESL27D variants altered GroEL ATP hydrolysis significantly (supplemental Fig.…”

Section: Resultsmentioning

confidence: 99%

Stimulating the Substrate Folding Activity of a Single Ring GroEL Variant by Modulating the Cochaperonin GroES

Illingworth

Ramsey

Zheng

2011

Journal of Biological Chemistry

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In mediating protein folding, chaperonin GroEL and cochaperonin GroES form an enclosed chamber for substrate proteins in an ATP-dependent manner. The essential role of the double ring assembly of GroEL is demonstrated by the functional deficiency of the single ring GroEL SR . The GroEL SR -GroES is highly stable with minimal ATPase activity. To restore the ATP cycle and the turnover of the folding chamber, we sought to weaken the GroEL SR -GroES interaction systematically by concatenating seven copies of groES to generate groES 7 . GroES Ile-25, Val-26, and Leu-27, residues on the GroEL-GroES interface, were substituted with Asp on different groES modules of groES 7 . GroES 7

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Effect of the C-terminal Truncation on the Functional Cycle of Chaperonin GroEL

Cited by 30 publications

References 35 publications

Substrate protein dependence of GroEL–GroES interaction cycle revealed by high-speed atomic force microscopy imaging

Substrate protein dependence of GroEL–GroES interaction cycle revealed by high-speed atomic force microscopy imaging

Single-molecule Study on the Decay Process of the Football-shaped GroEL-GroES Complex Using Zero-mode Waveguides

Stimulating the Substrate Folding Activity of a Single Ring GroEL Variant by Modulating the Cochaperonin GroES

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