Triggering Protein Folding within the GroEL-GroES Complex

Madan, Damian; Lin, Zong; Rye, Hays S.

doi:10.1074/jbc.m802898200

Cited by 35 publications

(66 citation statements)

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“…The functional dependence of the two fractions on ATP concentration suggests a cooperative process. Indeed, it was possible to fit the two sets of fractions to a Hill-type function with a Hill coefficient of 2.7 AE 0.1, which is similar to values obtained by other very different methods (14)(15)(16). Most importantly, even at the highest concentration of ATP the fraction of molecules in the Tstate is not 0 but 0.53 AE 0.02.…”

Section: Frank Et Almentioning

confidence: 49%

“…In kinesin, ADP release is again the trigger that resets the kinetic cycle, causing it to be ultraslow in the absence of microtubules and much faster when microtubules are available (14). The slow release of ADP from the trans ring of GroEL (i.e., the ring without bound GroES) is known from kinetic studies to time the cycle of the chaperone by effectively modulating the interaction between the two rings (15,16). Such slow ADP dissociation has the effects that (i) folding in the cis cavity can continue for a longer period of time and that (ii) the trans ring has ample time to bind a protein substrate and, thus, initiate a new reaction cycle.…”

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confidence: 99%

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Out-of-equilibrium conformational cycling of GroEL under saturating ATP concentrations

Frank

Goomanovsky

Davidi³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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The molecular chaperone GroEL exists in at least two allosteric states, T and R, that interconvert in an ATP-controlled manner. Thermodynamic analysis suggests that the T-state population becomes negligible with increasing ATP concentrations, in conflict with the requirement for conformational cycling, which is essential for the operation of molecular machines. To solve this conundrum, we performed fluorescence correlation spectroscopy on the single-ring version of GroEL, using a fluorescent switch recently built into its structure, which turns "on," i.e., increases its fluorescence dramatically, when ATP is added. A series of correlation functions was measured as a function of ATP concentration and analyzed using singular-value decomposition. The analysis assigned the signal to two states whose dynamics clearly differ. Surprisingly, even at ATP saturation, ∼50% of the molecules still populate the T state at any instance of time, indicating constant out-of-equilibrium cycling between T and R. Only upon addition of the cochaperonin GroES does the T-state population vanish. Our results suggest a model in which the T/R ratio is controlled by the rate of ADP release after hydrolysis, which can be determined accordingly.allostery | conformational dynamics | fluorescence correlation spectroscopy | molecular chaperones | chaperonins A TP-driven protein machines are abundant and contribute to multiple essential biological processes (1). Such protein machines undergo motor-like rotational motion (like F 1 -ATPase), carry cargos (like kinesin), or fold proteins (like GroEL, the subject of this paper). An important feature of all ATP-driven molecular machines is a functional cycle that involves sequential transitions between several conformational states (2). Understanding the dynamics of conformational cycling is, therefore, essential for the full elucidation of the mechanism of action of a protein machine.The Escherichia coli molecular chaperone GroEL is a machine that assists protein folding by undergoing a series of allosteric transitions that facilitate protein substrate binding and release (3,4). GroEL is made up of two homoheptameric rings, stacked back-to-back, with a cavity at each end (5), in which protein folding can take place. The allosteric transitions of GroEL are induced by ATP binding that occurs with positive cooperativity within rings and negative cooperativity between rings (6). It has been suggested that the intraring positive cooperativity is an outcome of a concerted switch between two conformations, Tand R, with low and high affinities for ATP, respectively. GroEL functions in conjunction with a heptameric ring-shaped cochaperonin, GroES. Binding of GroES to the so-called cis ring induces an additional conformational change that leads to the R' conformation and triggers dissociation of bound protein substrate into the cavity (7). The structures of the three relatively stable conformations, T, R, and R', have been determined using x-ray crystallography and electron microscopy (8, 9). It is not known ...

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Section: Frank Et Almentioning

confidence: 49%

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confidence: 99%

Out-of-equilibrium conformational cycling of GroEL under saturating ATP concentrations

Frank

Goomanovsky

Davidi³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…Folding rates (and the yield of in-cage folding) vary depending on the combination of chaperonin variants and substrate proteins (9,15), and the mutational effect of chaperonin on protein folding is not straightforward. Hydrophobicity of the cage wall has been thought to be an important factor that affects folding, but both the less hydrophobic SR398(Y203C) and the more hydrophobic pyrene-labeled SR398(F44C) fold rhodanese more slowly than SR398.…”

Section: Discussionmentioning

confidence: 99%

Revisiting the contribution of negative charges on the chaperonin cage wall to the acceleration of protein folding

Motojima

Motojima-Miyazaki

Yoshida

2012

Proc. Natl. Acad. Sci. U.S.A.

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Chaperonin GroEL mediates the folding of protein encapsulated in a GroES-sealed cavity (cage). Recently, a critical role of negative charge clusters on the cage wall in folding acceleration was proposed based on experiments using GroEL single-ring (SR) mutants SR1 and SRKKK2 [Tang YC, et al. (2006) Cell 125:903-914; Chakraborty K, et al. (2010) Cell 142:112-122]. Here, we revisited these experiments and discovered several inconsistencies. (i) SR1 was assumed to bind to GroES stably and to mediate single-round folding in the cage. However, we show that SR1 repeats multiple turnovers of GroES release/binding coupled with ATP hydrolysis.(ii) Although the slow folding observed for a double-mutant of maltose binding protein (DMMBP) by SRKKK2 was attributed to mutations that neutralize negative charges on the cage wall, we found that the majority of DMMBP escape from SRKKK2 and undergo spontaneous folding in the bulk medium. (iii) An osmolyte, trimethylamine N-oxide, was reported to accelerate SRKKK2-mediated folding of DMMBP by mimicking the effect of cage-wall negative charges of WT GroEL and ordering the water structure to promote protein compaction. However, we demonstrate that in-cage folding by SRKKK2 is unaffected by trimethylamine N-oxide. (iv) Although it was reported that SRKKK2 lost the ability to assist the folding of ribulose-1,5-bisphosphate carboxylase/oxygenase, we found that SRKKK2 retains this ability. Our results argue against the role of the negative charges on the cage wall of GroEL in protein folding. Thus, in chaperonin studies, folding kinetics need to be determined from the fraction of the real in-cage folding.guanidium chloride | urea | fluorescence | anisotropy | apyrase T he bacterial GroEL/GroES chaperonin is an essential molecular chaperone that mediates the folding of various proteins (1, 2). GroEL consists of two rings stacked back to back, and each ring, made up of seven 57-kDa subunits, possesses a large central cavity. GroEL binds to a wide range of denatured proteins at the hydrophobic apical end of the central cavity to make a binary complex of GroEL/substrate protein. On binding of ATP to GroEL, GroES attaches to the apical end of the GroEL ring as a lid, generating a GroEL/GroES/substrate protein ternary complex, in which the substrate protein in the sealed cage starts folding. Hydrolysis of bound ATP triggers the detachment of the GroES lid to allow the substrate protein in the cage, whether folded or denatured, to be free. The next denatured protein is captured by GroEL, ATP binds to GroEL, and the cycle repeats. Single-round folding can be observed without the complication of coordinated ATP hydrolysis turnover of the two rings in GroEL by the use of a single-ring (SR) mutant of GroEL (SR1) that holds the GroES lid long enough for the substrate protein to finish folding in the cage (3).Two model mechanisms for the function of chaperonin in mediating protein folding have been proposed. The passive Anfinsen cage model explains that proteins fold in a spontaneous manner in the c...

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“…Proteins-Wild-type and variants of GroEL (SR1, single-cysteine mutants and C-terminal truncation mutants), wild-type and E98C GroES, and wild-type and cysteine mutants of R. rubrum Rubisco were all expressed and purified as described previously (21)(22)(23)36).…”

Section: Methodsmentioning

confidence: 99%

The C-terminal Tails of the Bacterial Chaperonin GroEL Stimulate Protein Folding by Directly Altering the Conformation of a Substrate Protein

Weaver

Rye

2014

Journal of Biological Chemistry

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Background: Chaperonins like GroEL-GroES are required for the folding of many proteins. Results: The GroEL C termini alter substrate protein conformation and accelerate folding. Conclusion: Optimal protein folding requires the partial unfolding of misfolded states, a process that involves the GroEL C terminus. Significance: Chaperonins can actively facilitate protein folding by altering the conformations of folding intermediates.

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Triggering Protein Folding within the GroEL-GroES Complex

Cited by 35 publications

References 46 publications

Out-of-equilibrium conformational cycling of GroEL under saturating ATP concentrations

Out-of-equilibrium conformational cycling of GroEL under saturating ATP concentrations

Revisiting the contribution of negative charges on the chaperonin cage wall to the acceleration of protein folding

The C-terminal Tails of the Bacterial Chaperonin GroEL Stimulate Protein Folding by Directly Altering the Conformation of a Substrate Protein

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