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Takashi Funatsu - One of the best experts on this subject based on the ideXlab platform.
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chaperonin groel uses asymmetric and symmetric reaction cycles in response to the concentration of non native substrate proteins
Biophysics, 2016Co-Authors: Ryo Iizuka, Takashi FunatsuAbstract:: The Escherichia coli chaperonin GroEL is an essential molecular chaperone that mediates protein folding in association with its cofactor, GroES. It is widely accepted that GroEL alternates the GroES-sealed folding-active rings during the reaction cycle. In other words, an asymmetric GroEL-GroES complex is formed during the cycle, whereas a symmetric GroEL-(GroES)2 complex is not formed. However, this conventional view has been challenged by the recent reports indicating that such symmetric complexes can be formed in the GroEL-GroES reaction cycle. In this review, we discuss the studies of the symmetric GroEL-(GroES)2 complex, focusing on the molecular mechanism underlying its formation. We also suggest that GroEL can be involved in two types of reaction cycles (asymmetric or symmetric) and the type of cycle used depends on the concentration of non-native substrate proteins.
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single molecule study on the decay process of the football shaped groel GroES complex using zero mode waveguides
Journal of Biological Chemistry, 2010Co-Authors: Tomoya Sameshima, Mutsuko Aoki, Naonobu Shimamoto, Taro Ueno, Takashi Tanii, Iwao Ohdomari, Junichi Wada, Ryo Iizuka, Takashi FunatsuAbstract: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 football-shaped 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.
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denatured proteins facilitate the formation of the football shaped groel GroES 2 complex
Biochemical Journal, 2010Co-Authors: Tomoya Sameshima, Taro Ueno, Ryo Iizuka, Takashi FunatsuAbstract:: Controversy exists over whether the chaperonin GroEL forms a GroEL-(GroES)2 complex (football-shaped complex) during its reaction cycle. We have revealed previously the existence of the football-shaped complex in the chaperonin reaction cycle using a FRET (fluorescence resonance energy transfer) assay [Sameshima, Ueno, Iizuka, Ishii, Terada, Okabe and Funatsu (2008) J. Biol. Chem. 283, 23765-23773]. Although denatured proteins alter the ATPase activity of GroEL and the dynamics of the GroEL-GroES interaction, the effect of denatured proteins on the formation of the football-shaped complex has not been characterized. In the present study, a FRET assay was used to demonstrate that denatured proteins facilitate the formation of the football-shaped complex. The presence of denatured proteins was also found to increase the rate of association of GroES to the trans-ring of GroEL. Furthermore, denatured proteins decrease the inhibitory influence of ADP on ATP-induced association of GroES to the trans-ring of GroEL. From these findings we conclude that denatured proteins facilitate the dissociation of ADP from the trans-ring of GroEL and the concomitant association of ATP and the second GroES.
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Single-Molecule Imaging of 1:2 Groel-GroES Complexes in Zero-Mode Waveguides
Biophysical Journal, 2010Co-Authors: Tomoya Sameshima, Mutsuko Aoki, Naonobu Shimamoto, Taro Ueno, Takashi Tanii, Iwao Ohdomari, Junichi Wada, Ryo Iizuka, Takashi FunatsuAbstract:GroEL is an Escherichia coli chaperonin which is composed of two heptameric rings. GroEL interacts with its cofactor GroES and assists protein folding in an ATP dependent manner. Because of negative cooperativity between two rings of GroEL in the binding of ATP, it has been generally believed that an asymmetrical 1:1 complex is only a functional form for over a decade. Contrary to the belief, we revealed that a symmetrical 1:2 GroEL-GroES complex can be formed in the presence of denatured protein using fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS). However, the dynamics of GroEL-GroES interaction including a 1:2 GroEL-GroES complex is still unclear. To clarify this issue, 1:2 GroEL-GroES complexes were observed using zero-mode waveguides (ZMWs) at a single molecule level. ZMWs are nanoholes array fabricated in a thin metal film. They can reduce excitation volume compared to total internal reflection illumination; therefore, they make it possible to observe individual 1:2 GroEL-GroES complexes at sub-μM concentrations that are required to form the complexes. Cy3-GroES and Cy5-GroEL binding to and dissociating from Alexa488-GroES immobilized on the bottoms of ZMWs were visualized. Cy3-GroES and Cy5-GroEL were co-localized in GroES-immobilized ZMWs for ∼ 3 s. The duration time in ZMWs without immobilizing Alexa488-GroES, which reflected non-specific adsorption of GroEL-GroES complex to the bottoms of ZMWs, was ∼ 1 s. These results showed that 1:2 GroEL-GroES complexes were successfully observed at a single-molecule level and their duration time was estimated to be ∼3 s. Furthermore, the same experiment was carried out in the absence of denatured protein. The duration time of Cy3-GroES and Cy5-GroEL in Alexa488-GroES-immobilized ZMWs was ∼1 s. This result indicated that 1:2 GroEL-GroES complexes disappeared in the absence of denatured protein, being consistent with FRET and FCS experiments.
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football and bullet shaped groel GroES complexes coexist during the reaction cycle
Journal of Biological Chemistry, 2008Co-Authors: Tomoya Sameshima, Naofumi Terada, Kohki Okabe, Taro Ueno, Noriyuki Ishii, Ryo Iizuka, Takashi FunatsuAbstract:GroEL is an Escherichia coli chaperonin that is composed of two heptameric rings stacked back-to-back. GroEL assists protein folding with its cochaperonin GroES in an ATP-dependent manner in vitro and in vivo. However, it is still unclear whether GroES binds to both rings of GroEL simultaneously under physiological conditions. In this study, we monitored the GroEL-GroES interaction in the reaction cycle using fluorescence resonance energy transfer. We found that nearly equivalent amounts of symmetric GroEL-(GroES)2 (football-shaped) complex and asymmetric GroEL-GroES (bullet-shaped) complex coexist during the functional reaction cycle. We also found that D398A, an ATP hydrolysis defective mutant of GroEL, forms a football-shaped complex with ATP bound to the two rings. Furthermore, we showed that ADP prevents the association of ATP to the trans-ring of GroEL, and as a consequence, the second GroES cannot bind to GroEL. Considering the concentrations of ADP and ATP in E. coli, ADP is expected to have a small effect on the inhibition of GroES binding to the trans-ring of GroEL in vivo. These results suggest that we should reconsider the chaperonin-mediated protein-folding mechanism that involves the football-shaped complex.
Arthur L. Horwich - One of the best experts on this subject based on the ideXlab platform.
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a biochemical screen for groel GroES inhibitors
Bioorganic & Medicinal Chemistry Letters, 2014Co-Authors: Steven M. Johnson, Hsiao Ting Wang, Ingo H. Engels, Arthur L. Horwich, Orzala Sharif, Achim Brinker, Peter G Schultz, Eli ChapmanAbstract:Abstract High-throughput screening of 700,000 small molecules has identified 235 inhibitors of the GroEL/GroES-mediated refolding cycle. Dose–response analysis of a subset of these hits revealed that 21 compounds are potent inhibitors of GroEL/GroES-mediated refolding (IC50
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A biochemical screen for GroEL/GroES inhibitors
Bioorganic & Medicinal Chemistry Letters, 2014Co-Authors: Steven M. Johnson, Hsiao Ting Wang, Ingo H. Engels, Arthur L. Horwich, Orzala Sharif, Achim Brinker, Peter G Schultz, Eli ChapmanAbstract:High-throughput screening of 700,000 small molecules has identified 235 inhibitors of the GroEL/GroES-mediated refolding cycle. Dose–response analysis of a subset of these hits revealed that 21 compounds are potent inhibitors of GroEL/GroES-mediated refolding (IC50
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A biochemical screen for GroEL/GroES inhibitors
Bioorganic & Medicinal Chemistry Letters, 2014Co-Authors: Steven M. Johnson, Hsiao Ting Wang, Ingo H. Engels, Arthur L. Horwich, Orzala Sharif, Achim Brinker, Peter G Schultz, Eli ChapmanAbstract:High-throughput screening of 700,000 small molecules has identified 235 inhibitors of the GroEL/GroES-mediated refolding cycle. Dose–response analysis of a subset of these hits revealed that 21 compounds are potent inhibitors of GroEL/GroES-mediated refolding (IC50
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groel mediated protein folding
Protein Science, 2008Co-Authors: Wayne A Fenton, Arthur L. HorwichAbstract:I. Architecture of GroEL and GroES and the reaction pathway A. Architecture of the chaperonins B. Reaction pathway of GroEL-GroES-mediated folding II. Polypeptide binding A. A parallel network of chaperones binding polypeptides in vivo B. Polypeptide binding in vitro 1. Role of hydrophobicity in recognition 2. Homologous proteins with differing recognition-differences in primary structure versus effects on folding pathway 3. Conformations recognized by GroEL a. Refolding studies b. Binding of metastable intermediates c. Conformations while stably bound at GroEL 4. Binding constants and rates of association 5. Conformational changes in the substrate protein associated with binding by GroEL a. Observations b. Kinetic versus thermodynamic action of GroEL in mediating unfolding c. Crossing the energy landscape in the presence of GroEL III. ATP binding and hydrolysis-driving the reaction cycle IV. GroEL-GroES-polypeptide ternary complexes-the folding-active cis complex A. Cis and trans ternary complexes B. Symmetric complexes C. The folding-active intermediate of a chaperonin reaction-cis ternary complex D. The role of the cis space in the folding reaction E. Folding governed by a "timer" mechanism F. Release of nonnative polypeptides during the GroEL-GroES reaction G. Release of both native and nonnative forms under physiologic conditions H. A role for ATP binding, as well as hydrolysis, in the folding cycle V. Concluding remarks.
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exploring the structural dynamics of the e coli chaperonin groel using translation libration screw crystallographic refinement of intermediate states
Journal of Molecular Biology, 2004Co-Authors: Charu Chaudhry, Arthur L. Horwich, Axel T Brunger, Paul D. AdamsAbstract:Large rigid-body domain movements are critical to GroEL-mediated protein folding, especially apical domain elevation and twist associated with the formation of a folding chamber upon binding ATP and co-chaperonin GroES. Here, we have modeled the anisotropic displacements of GroEL domains from various crystallized states, unliganded GroEL, ATPγS-bound, ADP-AlFx/GroES-bound, and ADP/GroES bound, using translation-libration-screw (TLS) analysis. Remarkably, the TLS results show that the inherent motions of unliganded GroEL, a polypeptide-accepting state, are biased along the transition pathway that leads to the folding-active state. In the ADP-AlFx/GroES-bound folding-active state the dynamic modes of the apical domains become reoriented and coupled to the motions of bound GroES. The ADP/GroES complex exhibits these same motions, but they are increased in magnitude, potentially reflecting the decreased stability of the complex after nucleotide hydrolysis. Our results have allowed the visualization of the anisotropic molecular motions that link the static conformations previously observed by X-ray crystallography. Application of the same analyses to other macromolecules where rigid body motions occur may give insight into the large scale dynamics critical for function and thus has the potential to extend our fundamental understanding of molecular machines.
Taro Ueno - One of the best experts on this subject based on the ideXlab platform.
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single molecule study on the decay process of the football shaped groel GroES complex using zero mode waveguides
Journal of Biological Chemistry, 2010Co-Authors: Tomoya Sameshima, Mutsuko Aoki, Naonobu Shimamoto, Taro Ueno, Takashi Tanii, Iwao Ohdomari, Junichi Wada, Ryo Iizuka, Takashi FunatsuAbstract: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 football-shaped 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.
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denatured proteins facilitate the formation of the football shaped groel GroES 2 complex
Biochemical Journal, 2010Co-Authors: Tomoya Sameshima, Taro Ueno, Ryo Iizuka, Takashi FunatsuAbstract:: Controversy exists over whether the chaperonin GroEL forms a GroEL-(GroES)2 complex (football-shaped complex) during its reaction cycle. We have revealed previously the existence of the football-shaped complex in the chaperonin reaction cycle using a FRET (fluorescence resonance energy transfer) assay [Sameshima, Ueno, Iizuka, Ishii, Terada, Okabe and Funatsu (2008) J. Biol. Chem. 283, 23765-23773]. Although denatured proteins alter the ATPase activity of GroEL and the dynamics of the GroEL-GroES interaction, the effect of denatured proteins on the formation of the football-shaped complex has not been characterized. In the present study, a FRET assay was used to demonstrate that denatured proteins facilitate the formation of the football-shaped complex. The presence of denatured proteins was also found to increase the rate of association of GroES to the trans-ring of GroEL. Furthermore, denatured proteins decrease the inhibitory influence of ADP on ATP-induced association of GroES to the trans-ring of GroEL. From these findings we conclude that denatured proteins facilitate the dissociation of ADP from the trans-ring of GroEL and the concomitant association of ATP and the second GroES.
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Single-Molecule Imaging of 1:2 Groel-GroES Complexes in Zero-Mode Waveguides
Biophysical Journal, 2010Co-Authors: Tomoya Sameshima, Mutsuko Aoki, Naonobu Shimamoto, Taro Ueno, Takashi Tanii, Iwao Ohdomari, Junichi Wada, Ryo Iizuka, Takashi FunatsuAbstract:GroEL is an Escherichia coli chaperonin which is composed of two heptameric rings. GroEL interacts with its cofactor GroES and assists protein folding in an ATP dependent manner. Because of negative cooperativity between two rings of GroEL in the binding of ATP, it has been generally believed that an asymmetrical 1:1 complex is only a functional form for over a decade. Contrary to the belief, we revealed that a symmetrical 1:2 GroEL-GroES complex can be formed in the presence of denatured protein using fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS). However, the dynamics of GroEL-GroES interaction including a 1:2 GroEL-GroES complex is still unclear. To clarify this issue, 1:2 GroEL-GroES complexes were observed using zero-mode waveguides (ZMWs) at a single molecule level. ZMWs are nanoholes array fabricated in a thin metal film. They can reduce excitation volume compared to total internal reflection illumination; therefore, they make it possible to observe individual 1:2 GroEL-GroES complexes at sub-μM concentrations that are required to form the complexes. Cy3-GroES and Cy5-GroEL binding to and dissociating from Alexa488-GroES immobilized on the bottoms of ZMWs were visualized. Cy3-GroES and Cy5-GroEL were co-localized in GroES-immobilized ZMWs for ∼ 3 s. The duration time in ZMWs without immobilizing Alexa488-GroES, which reflected non-specific adsorption of GroEL-GroES complex to the bottoms of ZMWs, was ∼ 1 s. These results showed that 1:2 GroEL-GroES complexes were successfully observed at a single-molecule level and their duration time was estimated to be ∼3 s. Furthermore, the same experiment was carried out in the absence of denatured protein. The duration time of Cy3-GroES and Cy5-GroEL in Alexa488-GroES-immobilized ZMWs was ∼1 s. This result indicated that 1:2 GroEL-GroES complexes disappeared in the absence of denatured protein, being consistent with FRET and FCS experiments.
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football and bullet shaped groel GroES complexes coexist during the reaction cycle
Journal of Biological Chemistry, 2008Co-Authors: Tomoya Sameshima, Naofumi Terada, Kohki Okabe, Taro Ueno, Noriyuki Ishii, Ryo Iizuka, Takashi FunatsuAbstract:GroEL is an Escherichia coli chaperonin that is composed of two heptameric rings stacked back-to-back. GroEL assists protein folding with its cochaperonin GroES in an ATP-dependent manner in vitro and in vivo. However, it is still unclear whether GroES binds to both rings of GroEL simultaneously under physiological conditions. In this study, we monitored the GroEL-GroES interaction in the reaction cycle using fluorescence resonance energy transfer. We found that nearly equivalent amounts of symmetric GroEL-(GroES)2 (football-shaped) complex and asymmetric GroEL-GroES (bullet-shaped) complex coexist during the functional reaction cycle. We also found that D398A, an ATP hydrolysis defective mutant of GroEL, forms a football-shaped complex with ATP bound to the two rings. Furthermore, we showed that ADP prevents the association of ATP to the trans-ring of GroEL, and as a consequence, the second GroES cannot bind to GroEL. Considering the concentrations of ADP and ATP in E. coli, ADP is expected to have a small effect on the inhibition of GroES binding to the trans-ring of GroEL in vivo. These results suggest that we should reconsider the chaperonin-mediated protein-folding mechanism that involves the football-shaped complex.
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groel mediates protein folding with a two successive timer mechanism
Molecular Cell, 2004Co-Authors: Taro Ueno, Hisashi Tadakuma, Hideki Taguchi, Masasuke Yoshida, Takashi FunatsuAbstract:GroEL encapsulates nonnative substrate proteins in a central cavity capped by GroES, providing a safe folding cage. Conventional models assume that a single timer lasting ∼8 s governs the ATP hydrolysis-driven GroEL chaperonin cycle. We examine single molecule imaging of GFP folding within the cavity, binding release dynamics of GroEL-GroES, ensemble measurements of GroEL/substrate FRET, and the initial kinetics of GroEL ATPase activity. We conclude that the cycle consists of two successive timers of ∼3 s and ∼5 s duration. During the first timer, GroEL is bound to ATP, substrate protein, and GroES. When the first timer ends, the substrate protein is released into the central cavity and folding begins. ATP hydrolysis and phosphate release immediately follow this transition. ADP, GroES, and substrate depart GroEL after the second timer is complete. This mechanism explains how GroES binding to a GroEL-substrate complex encapsulates the substrate rather than allowing it to escape into solution.
Ryo Iizuka - One of the best experts on this subject based on the ideXlab platform.
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chaperonin groel uses asymmetric and symmetric reaction cycles in response to the concentration of non native substrate proteins
Biophysics, 2016Co-Authors: Ryo Iizuka, Takashi FunatsuAbstract:: The Escherichia coli chaperonin GroEL is an essential molecular chaperone that mediates protein folding in association with its cofactor, GroES. It is widely accepted that GroEL alternates the GroES-sealed folding-active rings during the reaction cycle. In other words, an asymmetric GroEL-GroES complex is formed during the cycle, whereas a symmetric GroEL-(GroES)2 complex is not formed. However, this conventional view has been challenged by the recent reports indicating that such symmetric complexes can be formed in the GroEL-GroES reaction cycle. In this review, we discuss the studies of the symmetric GroEL-(GroES)2 complex, focusing on the molecular mechanism underlying its formation. We also suggest that GroEL can be involved in two types of reaction cycles (asymmetric or symmetric) and the type of cycle used depends on the concentration of non-native substrate proteins.
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single molecule study on the decay process of the football shaped groel GroES complex using zero mode waveguides
Journal of Biological Chemistry, 2010Co-Authors: Tomoya Sameshima, Mutsuko Aoki, Naonobu Shimamoto, Taro Ueno, Takashi Tanii, Iwao Ohdomari, Junichi Wada, Ryo Iizuka, Takashi FunatsuAbstract: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 football-shaped 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.
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denatured proteins facilitate the formation of the football shaped groel GroES 2 complex
Biochemical Journal, 2010Co-Authors: Tomoya Sameshima, Taro Ueno, Ryo Iizuka, Takashi FunatsuAbstract:: Controversy exists over whether the chaperonin GroEL forms a GroEL-(GroES)2 complex (football-shaped complex) during its reaction cycle. We have revealed previously the existence of the football-shaped complex in the chaperonin reaction cycle using a FRET (fluorescence resonance energy transfer) assay [Sameshima, Ueno, Iizuka, Ishii, Terada, Okabe and Funatsu (2008) J. Biol. Chem. 283, 23765-23773]. Although denatured proteins alter the ATPase activity of GroEL and the dynamics of the GroEL-GroES interaction, the effect of denatured proteins on the formation of the football-shaped complex has not been characterized. In the present study, a FRET assay was used to demonstrate that denatured proteins facilitate the formation of the football-shaped complex. The presence of denatured proteins was also found to increase the rate of association of GroES to the trans-ring of GroEL. Furthermore, denatured proteins decrease the inhibitory influence of ADP on ATP-induced association of GroES to the trans-ring of GroEL. From these findings we conclude that denatured proteins facilitate the dissociation of ADP from the trans-ring of GroEL and the concomitant association of ATP and the second GroES.
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Single-Molecule Imaging of 1:2 Groel-GroES Complexes in Zero-Mode Waveguides
Biophysical Journal, 2010Co-Authors: Tomoya Sameshima, Mutsuko Aoki, Naonobu Shimamoto, Taro Ueno, Takashi Tanii, Iwao Ohdomari, Junichi Wada, Ryo Iizuka, Takashi FunatsuAbstract:GroEL is an Escherichia coli chaperonin which is composed of two heptameric rings. GroEL interacts with its cofactor GroES and assists protein folding in an ATP dependent manner. Because of negative cooperativity between two rings of GroEL in the binding of ATP, it has been generally believed that an asymmetrical 1:1 complex is only a functional form for over a decade. Contrary to the belief, we revealed that a symmetrical 1:2 GroEL-GroES complex can be formed in the presence of denatured protein using fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS). However, the dynamics of GroEL-GroES interaction including a 1:2 GroEL-GroES complex is still unclear. To clarify this issue, 1:2 GroEL-GroES complexes were observed using zero-mode waveguides (ZMWs) at a single molecule level. ZMWs are nanoholes array fabricated in a thin metal film. They can reduce excitation volume compared to total internal reflection illumination; therefore, they make it possible to observe individual 1:2 GroEL-GroES complexes at sub-μM concentrations that are required to form the complexes. Cy3-GroES and Cy5-GroEL binding to and dissociating from Alexa488-GroES immobilized on the bottoms of ZMWs were visualized. Cy3-GroES and Cy5-GroEL were co-localized in GroES-immobilized ZMWs for ∼ 3 s. The duration time in ZMWs without immobilizing Alexa488-GroES, which reflected non-specific adsorption of GroEL-GroES complex to the bottoms of ZMWs, was ∼ 1 s. These results showed that 1:2 GroEL-GroES complexes were successfully observed at a single-molecule level and their duration time was estimated to be ∼3 s. Furthermore, the same experiment was carried out in the absence of denatured protein. The duration time of Cy3-GroES and Cy5-GroEL in Alexa488-GroES-immobilized ZMWs was ∼1 s. This result indicated that 1:2 GroEL-GroES complexes disappeared in the absence of denatured protein, being consistent with FRET and FCS experiments.
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football and bullet shaped groel GroES complexes coexist during the reaction cycle
Journal of Biological Chemistry, 2008Co-Authors: Tomoya Sameshima, Naofumi Terada, Kohki Okabe, Taro Ueno, Noriyuki Ishii, Ryo Iizuka, Takashi FunatsuAbstract:GroEL is an Escherichia coli chaperonin that is composed of two heptameric rings stacked back-to-back. GroEL assists protein folding with its cochaperonin GroES in an ATP-dependent manner in vitro and in vivo. However, it is still unclear whether GroES binds to both rings of GroEL simultaneously under physiological conditions. In this study, we monitored the GroEL-GroES interaction in the reaction cycle using fluorescence resonance energy transfer. We found that nearly equivalent amounts of symmetric GroEL-(GroES)2 (football-shaped) complex and asymmetric GroEL-GroES (bullet-shaped) complex coexist during the functional reaction cycle. We also found that D398A, an ATP hydrolysis defective mutant of GroEL, forms a football-shaped complex with ATP bound to the two rings. Furthermore, we showed that ADP prevents the association of ATP to the trans-ring of GroEL, and as a consequence, the second GroES cannot bind to GroEL. Considering the concentrations of ADP and ATP in E. coli, ADP is expected to have a small effect on the inhibition of GroES binding to the trans-ring of GroEL in vivo. These results suggest that we should reconsider the chaperonin-mediated protein-folding mechanism that involves the football-shaped complex.
Damian Madan - One of the best experts on this subject based on the ideXlab platform.
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visualizing groel es in the act of encapsulating a folding protein
Cell, 2013Co-Authors: Donghua Chen, Damian Madan, Jeremy Weaver, Gunnar F Schroder, Wah ChiuAbstract:The GroEL/ES chaperonin system is required for the assisted folding of many proteins. How these substrate proteins are encapsulated within the GroEL-GroES cavity is poorly understood. Using symmetry-free, single-particle cryo-electron microscopy, we have characterized a chemically modified mutant of GroEL (EL43Py) that is trapped at a normally transient stage of substrate protein encapsulation. We show that the symmetric pattern of the GroEL subunits is broken as the GroEL cis-ring apical domains reorient to accommodate the simultaneous binding of GroES and an incompletely folded substrate protein (RuBisCO). The collapsed RuBisCO folding intermediate binds to the lower segment of two apical domains, as well as to the normally unstructured GroEL C-terminal tails. A comparative structural analysis suggests that the allosteric transitions leading to substrate protein release and folding involve concerted shifts of GroES and the GroEL apical domains and C-terminal tails.
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Visualizing Groel/Es in the Act of Encapsulating a Folding Protein
Cell, 2013Co-Authors: Donghua Chen, Damian Madan, Jeremy Weaver, Gunnar F Schroder, Wah ChiuAbstract:The GroEL/ES chaperonin system is required for the assisted folding of many proteins. How these substrate proteins are encapsulated within the GroEL-GroES cavity is poorly understood. Using symmetry-free, single-particle cryo-electron microscopy, we have characterized a chemically modified mutant of GroEL (EL43Py) that is trapped at a normally transient stage of substrate protein encapsulation. We show that the symmetric pattern of the GroEL subunits is broken as the GroEL cis-ring apical domains reorient to accommodate the simultaneous binding of GroES and an incompletely folded substrate protein (RuBisCO). The collapsed RuBisCO folding intermediate binds to the lower segment of two apical domains, as well as to the normally unstructured GroEL C-terminal tails. A comparative structural analysis suggests that the allosteric transitions leading to substrate protein release and folding involve concerted shifts of GroES and the GroEL apical domains and C-terminal tails.
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triggering protein folding within the groel GroES complex
Journal of Biological Chemistry, 2008Co-Authors: Damian MadanAbstract:The folding of many proteins depends on the assistance of chaperonins like GroEL and GroES and involves the enclosure of substrate proteins inside an internal cavity that is formed when GroES binds to GroEL in the presence of ATP. Precisely how assembly of the GroEL-GroES complex leads to substrate protein encapsulation and folding remains poorly understood. Here we use a chemically modified mutant of GroEL (EL43Py) to uncouple substrate protein encapsulation from release and folding. Although EL43Py correctly initiates a substrate protein encapsulation reaction, this mutant stalls in an intermediate allosteric state of the GroEL ring, which is essential for both GroES binding and the forced unfolding of the substrate protein. This intermediate conformation of the GroEL ring possesses simultaneously high affinity for both GroES and non-native substrate protein, thus preventing escape of the substrate protein while GroES binding and substrate protein compaction takes place. Strikingly, assembly of the folding-active GroEL-GroES complex appears to involve a strategic delay in ATP hydrolysis that is coupled to disassembly of the old, ADP-bound GroEL-GroES complex on the opposite ring.
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groel stimulates protein folding through forced unfolding
Nature Structural & Molecular Biology, 2008Co-Authors: Damian MadanAbstract:Many proteins cannot fold without the assistance of chaperonin machines like GroEL and GroES. The nature of this assistance, however, remains poorly understood. Here we demonstrate that unfolding of a substrate protein by GroEL enhances protein folding. We first show that capture of a protein on the open ring of a GroEL–ADP–GroES complex, GroEL's physiological acceptor state for non-native proteins in vivo, leaves the substrate protein in an unexpectedly compact state. Subsequent binding of ATP to the same GroEL ring causes rapid, forced unfolding of the substrate protein. Notably, the fraction of the substrate protein that commits to the native state following GroES binding and protein release into the GroEL–GroES cavity is proportional to the extent of substrate-protein unfolding. Forced protein unfolding is thus a central component of the multilayered stimulatory mechanism used by GroEL to drive protein folding.
