GroEL stimulates protein folding through forced unfolding

113

124

Molecular chaperones are known to be essential for avoiding protein aggregation in vivo, but it is still unclear how they affect protein folding mechanisms. We use single-molecule Förster resonance energy transfer to follow the folding of a protein inside the GroEL/GroES chaperonin cavity over a time range from milliseconds to hours. Our results show that confinement in the chaperonin decelerates the folding of the C-terminal domain in the substrate protein rhodanese, but leaves the folding rate of the N-terminal domain unaffected. Microfluidic mixing experiments indicate that strong interactions of the substrate with the cavity walls impede the folding process, but the folding hierarchy is preserved. Our results imply that no universal chaperonin mechanism exists. Rather, a competition between intra-and intermolecular interactions determines the folding rates and mechanisms of a substrate inside the GroEL/GroES cage.I n the recent past, a large number of components have been identified that control and modulate protein folding in vivo. This machinery includes molecular chaperones (1-3), sophisticated quality control systems, and complex mechanisms for protein translocation and degradation (3, 4), reflecting the importance of regulating the delicate balance of protein folding, misfolding, and aggregation in the cell. Such cellular factors exert conformational constraints on protein molecules that are expected to have a strong effect on the corresponding free-energy surfaces for folding (5). However, while the combination of cellular, biochemical, and structural data has led to some plausible qualitative models for the processes involved, mechanistic investigations comparable to those of autonomous protein folding in vitro (5-8) have been complicated by the complexity of the systems and the conformational heterogeneity involved (9). Even the autonomous folding of chaperone substrate proteins has been difficult to investigate because of their strong aggregation tendency (10). Contributions from confinement and crowding have been addressed in numerous studies using molecular simulations and theory (11)(12)(13)(14)(15)(16)(17)(18)(19)(20), but many of these concepts have eluded experimental examination.Here, we take a step towards closing this gap by investigating the GroEL/GroES chaperonin (1-3, 9) with single-molecule fluorescence spectroscopy (21-24), a method that is starting to provide previously inaccessible information on chaperonemediated protein folding (25)(26)(27)(28)(29)(30). GroEL/GroES is a remarkable molecular machine that binds nonnative proteins and allows them to fold within a cavity formed by the heptameric rings of GroEL and GroES. However, the cavity is only slightly larger than the folded structure of typical proteins known to interact with the chaperonin. The large volume of unconfined unfolded protein chains compared to the size of the cavity raises the question of whether and how such strong confinement affects the folding reaction (12-16, 18, 31, 32). By labeling the classic substrate prote...

Section: Resultsmentioning

confidence: 39%

Single-molecule spectroscopy of protein folding in a chaperonin cage

Hofmann

Hillger

Pfeil

et al. 2010

113

124

“…A number of studies have made it clear that the polypeptide binding step can rescue misfolded substrate proteins from kinetically trapped states that occur during folding (e.g., 4, 5), despite the lack of stable secondary structure in such conformations (6, 7). Such rescue has been associated with topological ''stretching'' of the substrate protein, as observed in a number of FRET studies (5,8,9). Substrate protein is released during large ATP/GroES-directed rigid body movements of the GroEL apical domains into the GroES-domed hydrophilic chamber, in which it proceeds to fold (10-12).…”

mentioning

confidence: 99%

“…Does polypeptide arrive first? This has been assumed in a number of studies in vitro, where an order of addition has been programmed in which substrate is first incubated with such asymmetrical complexes, followed by addition of ATP (8,9). In such studies, there have been observations that ATP addition produced additional stretching of nonnative protein, and it was suggested that this could constitute a necessary ATP-mediated ''forced unfolding'' step.…”

mentioning

confidence: 99%

GroEL/GroES cycling: ATP binds to an open ring before substrate protein favoring protein binding and production of the native state

Tyagi

Fenton

Horwich

2009

The GroEL/GroES reaction cycle involves steps of ATP and polypeptide binding to an open GroEL ring before the GroES encapsulation step that triggers productive folding in a sequestered chamber. The physiological order of addition of ATP and nonnative polypeptide, typically to the open trans ring of an asymmetrical GroEL/GroES/ ADP complex, has been unknown, although there have been assumptions that polypeptide binds first, allowing subsequent ATP-mediated movement of the GroEL apical domains to exert an action of forceful unfolding on the nonnative polypeptide. Here, using fluorescence measurements, we show that the physiological order of addition is the opposite, involving rapid binding of ATP, accompanied by nearly as rapid apical domain movements, followed by slower binding of nonnative polypeptide. In order-ofaddition experiments, approximately twice as much Rubisco activity was recovered when nonnative substrate protein was added after ATP compared with it being added before ATP, associated with twice as much Rubisco protein recovered with the chaperonin. Furthermore, the rate of Rubisco binding to an ATP-exposed ring was twice that observed in the absence of nucleotide. Finally, when both ATP and Rubisco were added simultaneously to a GroEL ring, simulating the physiological situation, the rate of Rubisco binding corresponded to that observed when ATP had been added first. We conclude that the physiological order, ATP binding before polypeptide, enables more efficient capture of nonnative substrate proteins, and thus allows greater recovery of the native state for any given round of the chaperonin cycle.chaperonin ͉ polypeptide binding ͉ protein folding T he GroEL/GroES chaperonin system provides assistance to folding of a large number of proteins to the native state via 2 principal actions, one involving binding of nonnative protein in an open ring of GroEL through multivalent hydrophobic contacts formed between the nonnative protein and surrounding GroEL apical domains and the other involving folding occurring in the encapsulated hydrophilic chamber formed when ATP and the GroES ''lid'' are bound to the same ring as polypeptide (1-3). A number of studies have made it clear that the polypeptide binding step can rescue misfolded substrate proteins from kinetically trapped states that occur during folding (e.g., 4, 5), despite the lack of stable secondary structure in such conformations (6, 7). Such rescue has been associated with topological ''stretching'' of the substrate protein, as observed in a number of FRET studies (5,8,9). Substrate protein is released during large ATP/GroES-directed rigid body movements of the GroEL apical domains into the GroES-domed hydrophilic chamber, in which it proceeds to fold (10-12). As shown in a number of studies, the so-called ''cis'' chamber facilitates folding by preventing multimolecular aggregation that could reduce both the rate of recovery of the native state (in those cases in which aggregation is reversible) and the extent of recovery (in those cases in which ag...

“…In particular, small ATP-directed elevation and twisting movements serve to recruit the cochaperonin lid structure, GroES (6). This triggers further large movements of the apical domains, Ϸ60°elevation and Ϸ120°clockwise twist, that release the substrate protein off the dislocating hydrophobic surface, effectively ejecting the substrate into a hydrophilic GroESencapsulated chamber where productive folding ensues (4,5,(7)(8)(9)(10)(11)(12)(13)(14). This step of ATP-mediated activation requires less than 1 s (9,12).…”

mentioning

confidence: 99%

Requirement for binding multiple ATPs to convert a GroEL ring to the folding-active state

Chapman

Farr

Fenton

et al. 2008

Production of the folding-active state of a GroEL ring involves initial cooperative binding of ATP, recruiting GroES, followed by large rigid body movements that are associated with ejection of bound substrate protein into the encapsulated hydrophilic chamber where folding commences. Here, we have addressed how many of the 7 subunits of a GroEL ring are required to bind ATP to drive these events, by using mixed rings with different numbers of wild-type and variant subunits, the latter bearing a substitution in the nucleotide pocket that allows specific block of ATP binding and turnover by a pyrazolol pyrimidine inhibitor. We observed that at least 2 wild-type subunits were required to bind GroES. By contrast, the triggering of polypeptide release and folding required a minimum of 4 wild-type subunits, with the greatest extent of refolding observed when all 7 subunits were wild type. This is consistent with the requirement for a ''power stroke'' of forceful apical movement to eject polypeptide into the chamber.chaperonin ͉ chemical inhibitor ͉ nucleotide ͉ protein folding ͉ pyrazolol pyrimidine