Chaperonin-mediated protein folding: using a central cavity to kinetically assist polypeptide chain folding

Horwich, Arthur L.; Fenton, Wayne A.

doi:10.1017/s0033583509004764

Cited by 135 publications

(137 citation statements)

References 103 publications

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“…This machine is ultimately powered by the binding and hydrolysis of ATP, driving GroEL through a series of allosteric states. Two views of GroEL function have emerged: one as a passive antiaggregation chamber and the other as an active folding device, performing work on SP (1,2). Our experiments support the latter view, demonstrating that GroEL has the ability to perform work on its SPs via domain movements that accompany the allosteric transitions.…”

supporting

confidence: 64%

Measuring how much work the chaperone GroEL can do

Corsepius

Lorimer

2013

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

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Noncovalently "stacked" tetramethylrhodamine (TMR) dimers have been used to both report and perturb the allosteric equilibrium in GroEL. A GroEL mutant (K242C) has been labeled with TMR, close to the peptide-binding site in the apical domain, such that TMR molecules on adjacent subunits are able to form dimers in the T allosteric state. Addition of ATP induces the transition to the R state and the separation of the peptide-binding sites, with concomitant unstacking of the TMR dimers. A statistical analysis of the spectra allowed us to compute the number and orientation of TMR dimers per ring as a function of the average number of TMR molecules per ring. The TMR dimers thus serve as quantitative reporter of the allosteric state of the system. The TMR dimers also serve as a surrogate for substrate protein, substituting in a more homogeneous, quantifiable manner for the heterogeneous intersubunit, intraring, noncovalent crosslinks provided by the substrate protein. The characteristic stimulation of the ATPase activity by substrate protein is also mimicked by the TMR dimers. Using an expanded version of the nested cooperativity model, we determine values for the free energy of the TT to TR and TR to RR allosteric equilibria to be 27 ± 11 and 46 ± 2 kJ/mol, respectively. The free energy of unstacking of the TMR dimers was estimated at 2.6 ± 1.0 kJ/mol dimer. These results demonstrate that GroEL can perform work during the T to R transition, supporting the iterative annealing model of chaperonin function.allostery | substrate protein binding problem

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supporting

confidence: 64%

Measuring how much work the chaperone GroEL can do

Corsepius

Lorimer

2013

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

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“…These proteins are found in all cell types in all organisms where they facilitate folding, assembly and transport. The most extensively studied chaperonin, both structurally and mechanistically, is the GroEL-GroES machinery (Horwich & Fenton, 2009). GroEL is a 780-kDA assembly consisting of two stacked cylindrical units, each of which is built up from seven identical 56-kDa subunits.…”

Section: Groelmentioning

confidence: 99%

“…GroEL is a 780-kDA assembly consisting of two stacked cylindrical units, each of which is built up from seven identical 56-kDa subunits. Encapsulation and release of substrate proteins is accomplished through a series of ATP-driven conformational changes to GroEL together with recruitment of the co-chaperone GroES (Horwich & Fenton, 2009). Questions under considerable debate in the scientific literature are: (1) how GroEL interacts with substrate proteins and (2) the mechanism of GroEL-mediated protein folding.…”

Section: Groelmentioning

confidence: 99%

Protein dynamics and function from solution state NMR spectroscopy

Kovermann

Rogne

Wolf‐Watz

2016

Quart. Rev. Biophys.

143

122

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Abstract. It is well-established that dynamics are central to protein function; their importance is implicitly acknowledged in the principles of the Monod, Wyman and Changeux model of binding cooperativity, which was originally proposed in 1965. Nowadays the concept of protein dynamics is formulated in terms of the energy landscape theory, which can be used to understand protein folding and conformational changes in proteins. Because protein dynamics are so important, a key to understanding protein function at the molecular level is to design experiments that allow their quantitative analysis. Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited for this purpose because major advances in theory, hardware, and experimental methods have made it possible to characterize protein dynamics at an unprecedented level of detail. Unique features of NMR include the ability to quantify dynamics (i) under equilibrium conditions without external perturbations, (ii) using many probes simultaneously, and (iii) over large time intervals. Here we review NMR techniques for quantifying protein dynamics on fast (ps-ns), slow (μs-ms), and very slow (s-min) time scales. These techniques are discussed with reference to some major discoveries in protein science that have been made possible by NMR spectroscopy.

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“…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.…”

mentioning

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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Chaperonin-mediated protein folding: using a central cavity to kinetically assist polypeptide chain folding

Cited by 135 publications

References 103 publications

Measuring how much work the chaperone GroEL can do

Measuring how much work the chaperone GroEL can do

Protein dynamics and function from solution state NMR spectroscopy

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

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