Stopped-flow Fluorescence Analysis of the Conformational Changes in the GroEL Apical Domain

Taniguchi, Masaaki; Yoshimi, Tatsunari; Hongo, Kunihiro; Mizobata, Tomohiro; Kawata, Yasushi

doi:10.1074/jbc.m311806200

Cited by 40 publications

(38 citation statements)

References 31 publications

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“…An additional kinetic study from Taniguchi et al (2004) monitored a tryptophan substituted at position 231 at one edge of the face of the apical domain, both in GroEL and SR1, observing the same results. These two studies thus offer parallel views of the steps taking place during the binding-to-folding transition of a ring.…”

Section: Fluorescence Changes Of Groel W485 and W231 As Monitors Of Ssupporting

confidence: 58%

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

Horwich

Fenton

2009

Quart. Rev. Biophys.

136

133

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The chaperonin ring assembly GroEL provides kinetic assistance to protein folding in the cell by binding non-native protein in the hydrophobic central cavity of an open ring and subsequently, upon binding ATP and the co-chaperonin GroES to the same ring, releasing polypeptide into a now hydrophilic encapsulated cavity where productive folding occurs in isolation. The fate of polypeptide during binding, encapsulation, and folding in the chamber has been the subject of recent experimental studies and is reviewed and considered here. We conclude that GroEL, in general, behaves passively with respect to its substrate proteins during these steps. While binding appears to be able to rescue non-native polypeptides from kinetic traps, such rescue is most likely exerted at the level of maximizing hydrophobic contact, effecting alteration of the topology of weakly structured states. Encapsulation does not appear to involve 'forced unfolding', and if anything, polypeptide topology is compacted during this step. Finally, chamber-mediated folding appears to resemble folding in solution, except that major kinetic complications of multimolecular association are prevented.

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Section: Fluorescence Changes Of Groel W485 and W231 As Monitors Of Ssupporting

confidence: 58%

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

Horwich

Fenton

2009

Quart. Rev. Biophys.

136

133

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“…When ATP binds rapidly to GroEL, does this cause significant adjustment in the apical polypeptide binding domains on the same rapid time scale, or are the apical changes in response to ATP binding relatively slow, such that the effects of ATP binding must be considered to be occurring at a later time? The earlier study of R231W had already indicated that apical effects occurred rapidly with that mutant (17). In the present study as well, a fluorescent probe on the outside aspect of the apical domains, remote from the ATP binding site on the inside of the cylinder (GroEL E315C in a cysteine-zero background labeled with OG), showed a rapid change in fluorescence intensity, on the same time scale as ATP binding.…”

Section: Equally Rapid Fluorescence Change On Atp Binding To Open Tramentioning

confidence: 48%

“…Moreover, polypeptide association with apical domains that have been mobilized could potentially affect the efficiency of binding. Such an order of addition with ATP binding before substrate protein seems plausible considering previous rate measurements that indicate, on one hand, rapid binding of ATP to unliganded GroEL (16)(17)(18) and, on the other hand, relatively slow binding of such substrate proteins as MDH and Rubisco to unoccupied GroEL or to asymmetrical GroEL/GroES/ADP complexes, respectively (19,20). Here, we have systematically investigated the relative rates of arrival of ATP and substrate protein ligands to both unliganded GroEL and asymmetrical GroEL/GroES/ADP complexes and find that the physiological order of arrival entails rapid ATP binding, producing nearly as rapid apical domain movement, followed by slower binding of substrate protein.…”

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confidence: 99%

“…1D), and a bimolecular rate constant of 2 ϫ 10 5 M Ϫ1 s Ϫ1 was determined for the faster phase. The slower kinetic phase, also dependent on ATP concentration, likely corresponds to the third fluorescent phase observed for GroEL's R231W and Y485W on ATP binding (17,18). The possibility that this phase reflected ATP hydrolysis was excluded by employing a hydrolysis-defective D398A/EL44-OG chaperonin.…”

mentioning

confidence: 99%

“…18; 123 Ϯ 2 s Ϫ1 ), and R231W (ref. 17; 80 s Ϫ1 ), where a tryptophan was substituted into the cavity-facing aspect of the apical domain (GroEL is otherwise devoid of tryptophan). As expected, the rate of interaction of ATP with EL44-OG was dependent on ATP concentration (Fig.…”

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confidence: 99%

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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

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

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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...

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ATP‐driven molecular chaperone machines

Clare

Saibil

2013

Biopolymers

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This review is focused on the mechanisms by which ATP binding and hydrolysis drive chaperone machines assisting protein folding and unfolding. A survey of the key, general chaperone systems Hsp70 and Hsp90, and the unfoldase Hsp100 is followed by a focus on the Hsp60 chaperonin machine which is understood in most detail. Cryo-electron microscopy analysis of the E. coli Hsp60 GroEL reveals intermediate conformations in the ATPase cycle and in substrate folding. These structures suggest a mechanism by which GroEL can forcefully unfold and then encapsulate substrates for subsequent folding in isolation from all other binding surfaces. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 846–859, 2013.

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Stopped-flow Fluorescence Analysis of the Conformational Changes in the GroEL Apical Domain

Cited by 40 publications

References 31 publications

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

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

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

ATP‐driven molecular chaperone machines

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