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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, Arthur L Horwich, Orzala Sharif, Hsiao Ting Wang, Ingo H Engels, 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, Arthur L Horwich, Orzala Sharif, Hsiao Ting Wang, Ingo H Engels, 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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atp triggered conformational changes delineate substrate binding and folding mechanics of the GroEL chaperonin
Cell, 2012Co-Authors: Daniel K. Clare, Arthur L Horwich, George W Farr, Daven Vasishtan, Scott M Stagg, Joel Quispe, Maya Topf, Helen R SaibilAbstract:Summary The chaperonin GroEL assists the folding of nascent or stress-denatured polypeptides by actions of binding and encapsulation. ATP binding initiates a series of conformational changes triggering the association of the cochaperonin GroES, followed by further large movements that eject the substrate polypeptide from hydrophobic binding sites into a GroES-capped, hydrophilic folding chamber. We used cryo-electron microscopy, statistical analysis, and flexible fitting to resolve a set of distinct GroEL-ATP conformations that can be ordered into a trajectory of domain rotation and elevation. The initial conformations are likely to be the ones that capture polypeptide substrate. Then the binding domains extend radially to separate from each other but maintain their binding surfaces facing the cavity, potentially exerting mechanical force upon kinetically trapped, misfolded substrates. The extended conformation also provides a potential docking site for GroES, to trigger the final, 100° domain rotation constituting the "power stroke" that ejects substrate into the folding chamber.
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double mutant mbp refolds at same rate in free solution as inside the GroEL groes chaperonin chamber when aggregation in free solution is prevented
FEBS Letters, 2011Co-Authors: Wayne A Fenton, Arthur L Horwich, Navneet K Tyagi, Ashok A DenizAbstract:Under “permissive” conditions at 25°C, the chaperonin substrate protein DM-MBP refolds 5–10 times more rapidly in the GroEL/GroES folding chamber than in free solution. This has been suggested to indicate that the chaperonin accelerates polypeptide folding by entropic effects of close confinement. Here, using native-purified DM-MBP, we show that the different rates of refolding are due to reversible aggregation of DM-MBP while folding free in solution, slowing its kinetics of renaturation: the protein exhibited concentration-dependent refolding in solution, with aggregation directly observed by dynamic light scattering. When refolded in chloride-free buffer, however, dynamic light scattering was eliminated, refolding became concentration-independent, and the rate of refolding became the same as that in GroEL/GroES. The GroEL/GroES chamber thus appears to function passively toward DM-MBP.
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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.
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 observation of protein folding in symmetric GroEL groes 2 complexes
Journal of Biological Chemistry, 2012Co-Authors: Yodai Takei, Ryo Iizuka, Taro Ueno, Takashi FunatsuAbstract:The chaperonin, GroEL, is an essential molecular chaperone that mediates protein folding together with its cofactor, GroES, in Escherichia coli. It is widely believed that the two rings of GroEL alternate between the folding active state coupled to GroES binding during the reaction cycle. In other words, an asymmetric GroEL-GroES complex (the bullet-shaped complex) is formed throughout the cycle, whereas a symmetric GroEL-(GroES)2 complex (the football-shaped complex) is not formed. We have recently shown that the football-shaped complex coexists with the bullet-shaped complex during the reaction cycle. However, how protein folding proceeds in the football-shaped complex remains poorly understood. Here, we used GFP as a substrate to visualize protein folding in the football-shaped complex by single-molecule fluorescence techniques. We directly showed that GFP folding occurs in both rings of the football-shaped complex. Remarkably, the folding was a sequential two-step reaction, and the kinetics were in excellent agreement with those in the bullet-shaped complex. These results demonstrate that the same reactions take place independently in both rings of the football-shaped complex to facilitate protein folding.
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Fabrication of zero-mode waveguide by ultraviolet nanoimprint lithography lift-off process
Japanese Journal of Applied Physics, 2011Co-Authors: Junichi Wada, Shou Ryu, Yuji Asano, Takao Yukawa, Takashi Funatsu, Taro Ueno, Jun Mizuno, Takashi TaniiAbstract:Zero-mode waveguides for single-molecule fluorescence imaging were fabricated using a simple desktop UV nanoimprint lithography system. An array of 30-to 150-nm-diameter nanoholes was successfully fabricated in an aluminum layer on a thin quartz plate by the single-step lift-off process using the UV-curable resist NIAC 707. Using the nanoholes, we performed real-time single-molecule fluorescence imaging to visualize the cochaperonin GroES binding with and dissociating from the chaperonin GroEL immobilized within the nanoholes. The demonstration revealed that the fluorescence from the GroES binding with the GroEL was three times stronger than the fluorescence from the GroES undergoing Brownian motion, and the real-time single-molecule fluorescence imaging was feasible using the zero-mode waveguide fabricated by the UV nanoimprint lithography lift-off process. © 2011 The Japan Society of Applied Physics.
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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, Junichi Wada, Takashi Tanii, Taro Ueno, Ryo Iizuka, Mutsuko Aoki, Naonobu Shimamoto, Iwao Ohdomari, 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.
Taro Ueno - One of the best experts on this subject based on the ideXlab platform.
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single molecule observation of protein folding in symmetric GroEL groes 2 complexes
Journal of Biological Chemistry, 2012Co-Authors: Yodai Takei, Ryo Iizuka, Taro Ueno, Takashi FunatsuAbstract:The chaperonin, GroEL, is an essential molecular chaperone that mediates protein folding together with its cofactor, GroES, in Escherichia coli. It is widely believed that the two rings of GroEL alternate between the folding active state coupled to GroES binding during the reaction cycle. In other words, an asymmetric GroEL-GroES complex (the bullet-shaped complex) is formed throughout the cycle, whereas a symmetric GroEL-(GroES)2 complex (the football-shaped complex) is not formed. We have recently shown that the football-shaped complex coexists with the bullet-shaped complex during the reaction cycle. However, how protein folding proceeds in the football-shaped complex remains poorly understood. Here, we used GFP as a substrate to visualize protein folding in the football-shaped complex by single-molecule fluorescence techniques. We directly showed that GFP folding occurs in both rings of the football-shaped complex. Remarkably, the folding was a sequential two-step reaction, and the kinetics were in excellent agreement with those in the bullet-shaped complex. These results demonstrate that the same reactions take place independently in both rings of the football-shaped complex to facilitate protein folding.
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Fabrication of zero-mode waveguide by ultraviolet nanoimprint lithography lift-off process
Japanese Journal of Applied Physics, 2011Co-Authors: Junichi Wada, Shou Ryu, Yuji Asano, Takao Yukawa, Takashi Funatsu, Taro Ueno, Jun Mizuno, Takashi TaniiAbstract:Zero-mode waveguides for single-molecule fluorescence imaging were fabricated using a simple desktop UV nanoimprint lithography system. An array of 30-to 150-nm-diameter nanoholes was successfully fabricated in an aluminum layer on a thin quartz plate by the single-step lift-off process using the UV-curable resist NIAC 707. Using the nanoholes, we performed real-time single-molecule fluorescence imaging to visualize the cochaperonin GroES binding with and dissociating from the chaperonin GroEL immobilized within the nanoholes. The demonstration revealed that the fluorescence from the GroES binding with the GroEL was three times stronger than the fluorescence from the GroES undergoing Brownian motion, and the real-time single-molecule fluorescence imaging was feasible using the zero-mode waveguide fabricated by the UV nanoimprint lithography lift-off process. © 2011 The Japan Society of Applied Physics.
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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, Junichi Wada, Takashi Tanii, Taro Ueno, Ryo Iizuka, Mutsuko Aoki, Naonobu Shimamoto, Iwao Ohdomari, 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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football and bullet shaped GroEL groes complexes coexist during the reaction cycle
Journal of Biological Chemistry, 2008Co-Authors: Tomoya Sameshima, Taro Ueno, Ryo Iizuka, Noriyuki Ishii, Naofumi Terada, Kohki Okabe, 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.
Hideki Taguchi - One of the best experts on this subject based on the ideXlab platform.
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Molecular Mechanism of Chaperonin GroEL: The Role of ATP and the Substrate Protein
Seibutsu Butsuri, 2020Co-Authors: Hideki TaguchiAbstract:The chaperonin GroEL is an essential molecular chaperone that assists protein folding in the cell. ATP-dependent conformational change of GroEL leads to the stable binding of cochaperonin GroES, forming a cage-shaped complex that accommodates a substrate protein to complete the folding. After the elucidation of the outline of the molecular mechanism over the last decade, now we are ready to answer the important questions; how GroEL encapsulate the substrate protein? How the substrate protein influences the functional cycle of GroEL? What is the role of ATP hydrolysis in the GroEL-assisted folding? Is the folding in the GroEL-ES cavity is same as that in the bulk solution? Here I review the recent progress on the GroEL study and discuss the essential role of chaperonin GroEL.
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effects of c terminal truncation of chaperonin GroEL on the yield of in cage folding of the green fluorescent protein
Journal of Biological Chemistry, 2015Co-Authors: So Ishino, Yasushi Kawata, Hideki Taguchi, Naoko Kajimura, Katsumi Matsuzaki, Masaru HoshinoAbstract:Abstract Chaperonin GroEL from Escherichia coli consists of two heptameric rings stacked back-to-back to form a cagelike structure. It assists in the folding of substrate proteins in concert with the co-chaperonin GroES by incorporating them into its large cavity. The mechanism underlying the incorporation of substrate proteins currently remains unclear. The flexible C-terminal residues of GroEL, which are invisible in the x-ray crystal structure, have recently been suggested to play a key role in the efficient encapsulation of substrates. These C-terminal regions have also been suggested to separate the double rings of GroEL at the bottom of the cavity. To elucidate the role of the C-terminal regions of GroEL on the efficient encapsulation of substrate proteins, we herein investigated the effects of C-terminal truncation on GroE-mediated folding using the green fluorescent protein (GFP) as a substrate. We demonstrated that the yield of in-cage folding mediated by a single ring GroEL (SR1) was markedly decreased by truncation, whereas that mediated by a double ring football-shaped complex was not affected. These results suggest that the C-terminal region of GroEL functions as a barrier between rings, preventing the leakage of GFP through the bottom space of the cage. We also found that once GFP folded into its native conformation within the cavity of SR1 it never escaped even in the absence of the C-terminal tails. This suggests that GFP molecules escaped through the pore only when they adopted a denatured conformation. Therefore, the folding and escape of GFP from C-terminally truncated SR1·GroES appeared to be competing with each other.
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difference in the distribution pattern of substrate enzymes in the metabolic network of escherichia coli according to chaperonin requirement
BMC Systems Biology, 2011Co-Authors: Kazuhiro Takemoto, Tatsuya Niwa, Hideki TaguchiAbstract:Background Chaperonins are important in living systems because they play a role in the folding of proteins. Earlier comprehensive analyses identified substrate proteins for which folding requires the chaperonin GroEL/GroES (GroE) in Escherichia coli, and they revealed that many chaperonin substrates are metabolic enzymes. This result implies the importance of chaperonins in metabolism. However, the relationship between chaperonins and metabolism is still unclear.
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revisiting the GroEL groes reaction cycle via the symmetric intermediate implied by novel aspects of the GroEL d398a mutant
Journal of Biological Chemistry, 2008Co-Authors: Ayumi Koiketakeshita, Masasuke Yoshida, Hideki TaguchiAbstract:The Escherichia coli chaperonin GroEL is a double-ring chaperone that assists in protein folding with the aid of GroES and ATP. It is believed that GroEL alternates the folding-active rings and that the substrate protein (and GroES) can bind to the open trans-ring only after ATP in the cis-ring is hydrolyzed. However, we found that a substrate protein prebound to the trans-ring remained bound during the first ATP cycle, and this substrate was assisted by GroEL-GroES when the second cycle began. Moreover, a slow ATP-hydrolyzing GroEL mutant (D398A) in the ATP-bound form bound a substrate protein and GroES to the trans-ring. The apparent discrepancy with the results from an earlier study (Rye, H. S., Roseman, A. M., Chen, S., Furtak, K., Fenton, W. A., Saibil, H. R., and Horwich, A. L. (1999) Cell 97, 325–338) can be explained by the previously unnoticed fact that the ATP-bound form of the D398A mutant exists as a symmetric 1:2 GroEL-GroES complex (the “football”-shaped complex) and that the substrate protein (and GroES) in the medium is incorporated into the complex only after the slow turnover. In light of these results, the current model of the GroEL-GroES reaction cycle via the asymmetric 1:1 GroEL-GroES complex deserves reexamination.
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Chaperonin GroEL Meets the Substrate Protein as a "Load" of the Rings
Journal of Biochemistry, 2005Co-Authors: Hideki TaguchiAbstract:Chaperonin GroEL is an essential molecular chaperone that assists protein folding in the cell. With the aid of cochaperonin GroES and ATP, double ring-shaped GroEL encapsulates non-native substrate proteins inside the cavity of the GroEL-ES complex. Although extensive studies have revealed the outline of GroEL mechanism over the past decade, central questions remain: What are the in vivo substrate proteins? How does GroEL encapsulate the substrates inside the cavity in spite of an apparent entropic difficulty? Is the folding inside the GroEL-ES cavity the same as bulk spontaneous folding? In this review I summarize the recent progress on in vivo and in vitro aspects of GroEL. In particular, emerging evidence shows that the substrate protein itself influences the chaperonin GroEL structure and reaction cycle. Finally I propose the mechanistic similarity between GroEL and kinesin, a molecular motor that moves along a microtubule in an ATP-dependent manner.
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 observation of protein folding in symmetric GroEL groes 2 complexes
Journal of Biological Chemistry, 2012Co-Authors: Yodai Takei, Ryo Iizuka, Taro Ueno, Takashi FunatsuAbstract:The chaperonin, GroEL, is an essential molecular chaperone that mediates protein folding together with its cofactor, GroES, in Escherichia coli. It is widely believed that the two rings of GroEL alternate between the folding active state coupled to GroES binding during the reaction cycle. In other words, an asymmetric GroEL-GroES complex (the bullet-shaped complex) is formed throughout the cycle, whereas a symmetric GroEL-(GroES)2 complex (the football-shaped complex) is not formed. We have recently shown that the football-shaped complex coexists with the bullet-shaped complex during the reaction cycle. However, how protein folding proceeds in the football-shaped complex remains poorly understood. Here, we used GFP as a substrate to visualize protein folding in the football-shaped complex by single-molecule fluorescence techniques. We directly showed that GFP folding occurs in both rings of the football-shaped complex. Remarkably, the folding was a sequential two-step reaction, and the kinetics were in excellent agreement with those in the bullet-shaped complex. These results demonstrate that the same reactions take place independently in both rings of the football-shaped complex to facilitate protein folding.
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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, Junichi Wada, Takashi Tanii, Taro Ueno, Ryo Iizuka, Mutsuko Aoki, Naonobu Shimamoto, Iwao Ohdomari, 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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football and bullet shaped GroEL groes complexes coexist during the reaction cycle
Journal of Biological Chemistry, 2008Co-Authors: Tomoya Sameshima, Taro Ueno, Ryo Iizuka, Noriyuki Ishii, Naofumi Terada, Kohki Okabe, 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.
