The complex kinetics of Pi and ADP release by the chaperonin GroEL/GroES is influenced by the presence of unfolded substrate protein (SP). Without SP, the kinetics of Pi release are described by four phases: a "lag," a "burst" of ATP hydrolysis by the nascent cis ring, a "delay" caused by ADP release from the nascent trans ring, and steady-state ATP hydrolysis. The release of Pi precedes the release of ADP. The rate-determining step of the asymmetric cycle is the release of ADP from the trans ring of the GroEL-GroES 1 "bullet" complex that is, consequently, the predominant species. In the asymmetric cycle, the two rings of GroEL function alternately, 180°out of phase. In the presence of SP, a change in the kinetic mechanism occurs. With SP present, the kinetics of ADP release are also described by four phases: a lag, a "surge" of ADP release attributable to SP-induced ADP/ATP exchange, and a "pause" during which symmetrical "football" particles are formed, followed by steady-state ATP hydrolysis. SP catalyzes ADP/ATP exchange on the trans ring. Now ADP release precedes the release of Pi, and the rate-determining step of the symmetric cycle becomes the hydrolysis of ATP by the symmetric GroEL-GroES 2 football complex that is, consequently, the predominant species. A FRET-based analysis confirms that asymmetric GroEL-GroES 1 bullets predominate in the absence of SP, whereas symmetric GroEL-GroES 2 footballs predominate in the presence of SP. This evidence suggests that symmetrical football particles are the folding functional form of the chaperonin machine in vivo.T he GroEL/GroES chaperonin machine is an indispensable, cellular device of exquisite complexity (1-3). Ultimately driven by the hydrolysis of ATP, it optimizes the folding of unfolded, client proteins under conditions where that thermodynamically favorable process does not occur (4). GroEL, the "engine," comprises two heptameric rings stacked back-to-back. Its subunits consist of equatorial, intermediate, and apical domains, which move in a concerted, rigid-body manner, swiveling on hinges located at the domain interfaces (5, 6). Each ring cycles through a progression of allosterically controlled conformational states in response to the binding of adenosine nucleotides and their associated metal ions, Mg 2+ and K + , the cochaperonin GroES, and, when present, the substrate protein (SP) (1-3). GroES functions like the lid on a cooking pot, transiently encapsulating a single molecule of SP, within the GroEL ring, the so-called Anfinsen cage (7). However, the SP does not remain encapsulated. Instead, with each chaperonin hemicycle, specifically in response to the binding of ATP to the opposite ring, first GroES and then SP is released, regardless of whether or not the SP has progressed to the folded state (8).Based mostly on studies of GroEL/GroES in the absence of SP, one of us (G.H.L.) likened its behavior to that of a twostroke motor (9). The two rings operate ∼180°out of phase with one another, hydrolyzing ATP alternately. The rate-determining step in ...

Using calibrated FRET, we show that the simultaneous occupancy of both rings of GroEL by ATP and GroES occurs, leading to the rapid formation of symmetric GroEL:GroES 2 "football" particles regardless of the presence or absence of substrate protein (SP). In the absence of SP, these symmetric particles revert to asymmetric GroEL:GroES 1 "bullet" particles. The breakage of GroES symmetry requires the stochastic hydrolysis of ATP and the breakage of nucleotide symmetry. These asymmetric particles are both persistent and dynamic; they turnover via the asymmetric cycle. When challenged with SP, however, they revert to symmetric particles within a second. In the presence of SP, the symmetric particles are also persistent and dynamic. They turn over via the symmetric cycle. Under these conditions, the stochastic hydrolysis of ATP and the breakage of nucleotide symmetry also occur within the ensemble of particles. However, on account of SP-catalyzed ADP/ATP exchange, GroES symmetry is rapidly restored. The residence time of both GroES and SP on functional GroEL is reduced to ∼1 s, enabling many more iterations than was previously believed possible, consistent with the iterative annealing mechanism. This result is inconsistent with currently accepted models. Using a foldable SP, we show that as the SP folds to the native state and the population of unfolded SP declines, the population of symmetric particles reverts to asymmetric particles in parallel, a result that is consistent with the former being the folding functional form.G roEL plays a central role in the function of the GroEL/ GroES nanomachine. It binds unfolded proteins, transiently encapsulates them under the GroES "lid," and then releases them to the external medium, this cycle of events being driven by the hydrolysis of ATP (1, 2). GroEL, however, consists of two heptameric rings, and some controversy has arisen as to whether each ring operates alternately via an asymmetric cycle or simultaneously via a symmetric cycle. In the former case, GroEL engages one GroES at a time and the predominant species is the asymmetric GroEL:GroES 1 "bullet" complex whereas, in the latter, symmetric GroEL:GroES 2 "football" particles predominate in the symmetric cycle (figure 9 in ref. 4) (3, 4). Nevertheless, the asymmetric cycle, championed by leading authorities (5, 6), has become the widely accepted model of chaperonin function, promulgated in recent textbooks (7, 8) despite much evidence (9-21) that assigns a role to the symmetric football particles. However, the involvement of these symmetric particles in chaperonin function has been questioned on the basis of three items of chaperonin dogma: (i) that the formation of symmetric GroEL:GroES 2 particles and polypeptide binding are mutually exclusive (22, 23); (ii) the belief that, because of negative cooperativity, "when one GroEL ring binds ATP, the other ring cannot also do so" (5); and (iii) that the chaperonin ATPase cycle turns over at the same rate in the presence of substrate protein as it does in its absence. Here...

The GroE chaperonins assist substrate protein (SP) folding by cycling through several conformational states. With each cycle the SP is, in turn, captured, unfolded, briefly encapsulated (t 1/2 ∼1 s), and released by the chaperonin complex. The protein-folding functional form is the US-football-shaped GroEL:GroES 2 complex. We report structures of two such "football" complexes to ∼3.7-Å resolution; one is empty whereas the other contains encapsulated SP in both chambers. Although encapsulated SP is not visible on the electron density map, using calibrated FRET and order-of-addition experiments we show that owing to SP-catalyzed ADP/ATP exchange both chambers of the football complex encapsulate SP efficiently only if the binding of SP precedes that of ATP. The two rings of GroEL thus behave as a parallel processing machine, rather than functioning alternately. Compared with the bullet-shaped GroEL:GroES 1 complex, the GroEL:GroES 2 football complex differs conformationally at the GroEL-GroES interface and also at the interface between the two GroEL rings. We propose that the electrostatic interactions between the e-NH 3+ of K105 of helix D in one ring with the negatively charged carboxyl oxygen of A109 at the carboxyl end of helix D of the other ring provide the structural basis for negative inter-ring cooperativity.symmetric | crystal structure | order-of-ligand-addition | encapsulation T he chaperonin proteins GroEL and GroES assist substrate proteins (SPs) to reach their native states, often under conditions when that otherwise spontaneous event does not occur (1-3). In the absence of SP GroEL/GroES operates via an asymmetric cycle in which the dissociation of ADP is the ratedetermining step and the predominant species is an asymmetric, bullet-shaped GroEL:GroES 1 complex (4, 5). The structure of this "bullet" GroEL:GroES 1 complex has long been known (6, 7) and it has been assumed that this is the species that assists protein folding (8, 9). However, there is much evidence for the involvement of symmetric, "football"-shaped GroEL:GroES 2 complexes (10-18). The formation of the football-shaped complex is promoted by SP (5, 17). Unfolded SP changes the kinetic mechanism, accelerating the rate of ADP/ATP exchange such that the dissociation of ADP is no longer rate-determining (Fig. 1A) (5). Thus, SP shifts the equilibrium between the footballs and bullets in favor of the former, consequently making them the predominant species (Fig. 1A) (5, 17).To elucidate the mechanism of chaperonin-assisted protein folding by the football complex, we investigated the conditions permitting the formation of the football complexes. Using calibrated FRET we show that owing to SP-catalyzed ADP/ATP exchange the football complex efficiently encapsulates SP in both GroEL chambers. Thus, the two rings of GroEL behave as a parallel processing machine, rather than functioning alternately. We also determined structures of two football complexes; one is empty, the other contains encapsulated SP in both chambers. However, encapsulated SP i...

Although native chemical ligation (NCL) and related chemoselective ligation approaches provide an elegant method to stitch together unprotected peptides, the handling and purification of insoluble and aggregation-prone peptides and assembly intermediates create a bottleneck to routinely preparing large proteins by completely synthetic means. In this work, we introduce a general new tool, Fmoc-Ddae-OH, N-Fmoc-1-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-3-[2-(2-aminoethoxy)ethoxy]-propan-1-ol, a hetero-bifunctional traceless linker for temporarily attaching highly solubilizing peptide sequences (helping hands) onto insoluble peptides. This tool is implemented in three simple and nearly quantitative steps: (i) on-resin incorporation of the linker at a Lys residue ε-amine, (ii) Fmoc-SPPS elongation of a desired solubilizing sequence, and (iii) in-solution removal of the solubilizing sequence using mild aqueous hydrazine to cleave the Ddae linker after NCL-based assembly. Successful introduction and removal of a Lys6 helping hand is first demonstrated in two model systems (Ebolavirus C20 peptide and the 70-residue ribosomal protein L31). It is then applied to the challenging chemical synthesis of the 97-residue co-chaperonin GroES, which contains a highly insoluble C-terminal segment that is rescued by a helping hand. Importantly, the Ddae linker can be cleaved in one pot following NCL or desulfurization. The purity, structure, and chaperone activity of synthetic L-GroES were validated with respect to a recombinant control. Additionally, the helping hand enabled synthesis of D-GroES, which was inactive in a hetero-chiral mixture with recombinant GroEL—providing additional insight into chaperone specificity. Ultimately, this simple, robust, and easy-to-use tool is expected to be broadly applicable for the synthesis of challenging peptides and proteins.

Hsp104 provides a valuable model for the many essential proteostatic functions performed by the AAA+ superfamily of protein molecular machines. We developed and used a powerful hydrogen exchange mass spectrometry (HX MS) analysis that can provide positionally resolved information on structure, dynamics, and energetics of the Hsp104 molecular machinery, even during functional cycling. HX MS reveals that the ATPase cycle is rate-limited by ADP release from nucleotide-binding domain 1 (NBD1). The middle domain (MD) serves to regulate Hsp104 activity by slowing ADP release. Mutational potentiation accelerates ADP release, thereby increasing ATPase activity. It reduces time in the open state, thereby decreasing substrate protein loss. During active cycling, Hsp104 transits repeatedly between whole hexamer closed and open states. Under diverse conditions, the shift of open/closed balance can lead to premature substrate loss, normal processing, or the generation of a strong pulling force. HX MS exposes the mechanisms of these functions at near-residue resolution.