Negative Cooperativity Research Articles

Novel nAChRs modulators for treating CNS disorders: discovery of AChBP cooperative ligands (1059.7)

nAChRs are pentameric molecules formed from identical or homologous subunits to form homomeric and heteromeric assemblies. They play a critical role in the peripheral and central nervous systems to mediate neurotransmission by acetylcholine and, as such, are critical targets for understanding nicotine addiction and developing useful drugs for treating CNS disorders such as schizophrenia, Parkinson’s disease and disorders of dementias. The acetylcholine binding protein (AChBP) is a pentameric structural homologue of the extracellular ligand‐binding domain of nAChRs. Up until recently, this approach has yielded compounds that bind as would be predicted by a equivalent set of five sites in the pentameric molecule, a situation also seen with classical natural product agonists and antagonists binding to the AChBP. Very recently through our synthetic program, we have uncovered a set of new structures that exhibit cooperativity, where many show Hill slopes greater than 1.0 or considerably less than 1.0. The latter is not due to heterogeneity of the binding protein template, since AChBP has five identical sites as examined crystallographically and by conventional ligand binding. Through the structural analysis, we can delineate the structural features of the ligands inducing this new mode of association with the extracellular domain of the receptor. Our approach of finding a series of ~50 congeneric ligands where individual members can be distinguished by positive and negative cooperativity should shed new light on subunit interactions in this homopentamer. For example, cooperative interactions appear to link in a circumferential fashion around the perimeter of the extracellular domain and do not necessarily require a direct connection with the channel gate area to elicit subunit interactions. This unique feature of our series of ligands gives a high promise to develop medications with distinct pharmacological profiles.Grant Funding Source: TRDRP (21FT_0024); NIH (GM 18360)

Role of human glutathione synthetase H‐loop residues in substrate binding and activity (773.4)

Glutathione (GSH) is an antioxidant essential for mammalian cell survival and is found in every cell of almost all organisms. GSH cannot be taken up by the cell, so understanding the intercellular synthesis of this important antioxidant is crucial. GSH is synthesized in a two‐step process. In the second step, human glutathione synthetase (hGS) binds γ‐glutamylcysteine and glycine and then uses the energy of ATP to make GSH. hGS is a homodimer that displays negative cooperativity, toward the γ‐glutamylcysteine substrate, a phenomenon which contributes to metabolic regulation but is not yet fully understood. The H‐loop (147‐TISASF‐152) of hGS is highly conserved and located near the binding site of γ‐glutamylcysteine. The role of H‐loop residues was examined using site‐directed mutagenesis of H‐loop residues, followed by purification and kinetic characterization of H‐loop mutant hGS enzymes. Our results show that the H‐loop mutant hGS enzymes have altered binding affinity (Km) for the negatively cooperative substrate, γ‐glutamylcysteine.Grant Funding Source: Supported by NIH R15GM086833 (MEA), REP Grant (TWU, MEA), Welch Chem Dept Grant (TWU)

Testing the versatility of the alternating sites of reactivity mechanism in the phosphagen kinases (768.11)

Phosphagen kinases achieve “buffering” of cellular ATP levels by catalyzing the reversible transfer of a phosphoryl group from ATP to a guanidino acceptor to form a high energy compound, phosphagen. Creatine kinase (CK), a PK found mainly in vertebrates, exists as a dimer in muscles; whereas, arginine kinase (AK), found exclusively in invertebrates, exists mainly as a monomer. The physiological functions of different oligomerization states within the PK family are not fully understood. Crystal structures of dimeric CK and AK are consistent with cooperativity in ligand binding between the two subunits. Based on these results, a reciprocating mechanism of catalysis was suggested for dimeric PKs. However, the exact mechanism and the potential effects of negative cooperativity on ligand binding ability of dimeric PKs remains unexplained. In this study, we’ve shown direct functional evidence of negative cooperativity in rabbit muscle CK (rbCK) by measuring the enzymes’ affinity for MgADP using isothermal titration calorimetry. Measurements of rbCK‐ligand interactions, in presence of saturating concentrations of creatine and nitrate, to form the transition‐state analogue quaternary complex, were fitted using a two‐site binding model with Kd1 = 56 ± 32 µM and Kd2 = 2.7 ± 1.4 mM. In the absence of creatine and nitrate, MgADP binding by rbCK fits a one‐site binding model, with Kd = 720 ± 330 µM. These suggest that the presence of creatine enables the conformation necessary to permit cross‐talk between the subunits. Pre‐steady state kinetic assays and the effect of viscosity on kcat show that the rate‐limiting step of reaction catalyzed by PKs is dependent on the oligomerization state of the enzyme, consistent with the alternating site of reactivity mechanism.Grant Funding Source: NSF Grant #0619123

Role of substrate loop residues in catalytic loop motion and substrate binding in human glutathione synthetase (773.2)

Human glutathione synthetase (hGS) catalyzes the second step in the synthesis of the key antioxidant glutathione. The homodimeric enzyme displays negative cooperativity towards the ɣ‐glutamylcysteine substrate. The presented work probes the significance of residues within the substrate binding loop of hGS responsible for loop motion throughout the catalytic cycle and provides insight into the precise mechanism of communication between active sites. Molecular dynamics simulations and docking studies elucidate the interactions that mediate binding. Experimental point mutations, coupled activity assays, kinetic studies, and differential scanning calorimetry provide information on the role of these residues in activity, cooperativity and thermal stability. A deeper understanding of the residues responsible for loop motion within the substrate binding loop of hGS shows that interactions at the active site have significant impact on the structure and function of the enzyme.Grant Funding Source: Supported by NIH R15GM086833, Research Enhancement Program Grant (TWU), UNT Faculty Research Grant

Characterization of Thermotoga maritima glycerol dehydrogenase for the enzymatic production of dihydroxyacetone

NAD-dependent Thermotoga maritima glycerol dehydrogenase (TmGlyDH) converts glycerol into dihydroxyacetone (DHA), a valuable synthetic precursor and sunless tanning agent. In this work, recombinant TmGlyDH was characterized to determine if it can be used to catalyze DHA production. The pH optima for glycerol oxidation and DHA reduction at 50°C were 7.9 and 6.0, respectively. Under the conditions tested, TmGlyDH had a linear Arrhenius plot up to 80°C. TmGlyDH was more thermostable than other glycerol dehydrogenases, remaining over 50% active after 7h at 50°C. TmGlyDH was active on racemic 1,2-propanediol and produced (R)-1,2-propanediol from hydroxyacetone with an enantiomeric excess above 99%, suggesting that TmGlyDH can also be used for chiral synthesis. (R)-1,2-propanediol production from hydroxyacetone was demonstrated for the first time in a one-enzyme cycling reaction using glycerol as the second substrate. Negative cooperativity was observed with glycerol and DHA, but not with the cofactor. Apparent kinetic parameters for glycerol, DHA, and NAD(H) were determined over a broad pH range. TmGlyDH showed little activity with N(6)-carboxymethyl-NAD(+) (N(6)-CM-NAD), an NAD(+) analog modified for easy immobilization to amino groups, but the double mutation V44A/K157G increased catalytic efficiency with N(6)-CM-NAD(+) ten-fold. Finally, we showed for the first time that a GlyDH is active with immobilized N(6)-CM-NAD(+), suggesting that N(6)-CM-NAD(+) can be immobilized on an electrode to allow TmGlyDH activity in a system that reoxidizes the cofactor electrocatalytically.

The two common polymorphic forms of human NRH-quinone oxidoreductase 2 (NQO2) have different biochemical properties.

There are two common forms of NRH-quinone oxidoreductase 2 (NQO2) in the human population resulting from SNP rs1143684. One has phenylalanine at position 47 (NQO2-F47) and the other leucine (NQO2-L47). Using recombinant proteins, we show that these variants have similar steady state kinetic parameters, although NQO2-L47 has a slightly lower specificity constant. NQO2-L47 is less stable towards proteolytic digestion and thermal denaturation than NQO2-F47. Both forms are inhibited by resveratrol, but NQO2-F47 shows negative cooperativity with this inhibitor. Thus these data demonstrate, for the first time, clear biochemical differences between the variants which help explain previous biomedical and epidemiological findings.

A B3LYP and MP2(full) theoretical investigation on the cooperativity effect between hydrogen-bonding and cation-molecule interactions and thermodynamic property in the 1: 2 (Na+: N-(Hydroxymethyl)acetamide) ternary complex

The cooperativity effects between the O/N-H∙∙∙O hydrogen-bonding and Na⁺∙∙∙O cation-molecule interactions in the 1: 2 (Na⁺: N-(Hydroxymethyl)acetamide) systems were investigated at the B3LYP/6-311++G**, MP2(full)/6-311++G** and MP2(full)/aug-cc-pvtz levels. The thermodynamic cooperativity calculations were also carried out for two pathways of the ternary-complex formation. The result shows that, in most ternary complexes, the O/N-H∙∙∙O and Na⁺∙∙∙O interactions are weakened in comparison with those in binary systems, leading to the anti-cooperativity effects, in particular in the complexes in which only the Na⁺∙∙∙O interactions exist. Shifts of electron density confirm the existence of anti-cooperativity. The increase of favorable enthalpic contribution leads to the positive cooperativity effect with negative ΔG(coop.) on forming the ternary complex by initial N-(Hydroxymethyl)acetamide dimer followed by addition of Na⁺. In forming the ternary complex by Na⁺∙∙∙N-(Hydroxymethyl)acetamide with the second N-(Hydroxymethyl)acetamide unit, the large unfavorable entropy change leads to the negative cooperativity effect with positive ΔG(coop.). The ternary complex is more easily formed by the pathway in which Na⁺ binds to N-(Hydroxymethyl)acetamide dimer.

Catalytic Site Cooperativity in Dimeric Methylthioadenosine Nucleosidase

5′-Methylthioadenosine/S-adenosylhomocysteine nucleosidases (MTANs) are bacterial enzymes that catalyze hydrolysis of the N-ribosidic bonds of 5′-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH) to form adenine and 5-thioribosyl groups. MTANs are involved in AI-1 and AI-2 bacterial quorum sensing and the unusual futalosine-based menaquinone synthetic pathway in Streptomyces,Helicobacter, and Campylobacter species. Crystal structures show MTANs to be homodimers with two catalytic sites near the dimer interface. Here, we explore the cooperative ligand interactions in the homodimer of Staphylococcus aureus MTAN (SaMTAN). Kinetic analysis indicated negative catalytic cooperativity. Titration of SaMTAN with the transition-state analogue MT-DADMe-ImmA gave unequal catalytic site binding, consistent with negative binding cooperativity. Thermodynamics of MT-DADMe-ImmA binding also gave negative cooperativity, where the first site had different enthalpic and entropic properties than the second site. Cysteine reactivity in a single-cysteine catalytic site loop construct of SaMTAN is reactive in native enzyme, less reactive when inhibitor is bound to one subunit, and nonreactive upon saturation with inhibitor. A fusion peptide heterodimer construct with one inactive subunit (E173Q) and one native subunit gave 25% of native SaMTAN activity, similar to native SaMTAN with MT-DADMe-ImmA at one catalytic site. Pre-steady-state kinetics showed fast chemistry at one catalytic site, consistent with slow adenine release before catalysis occurs at the second catalytic site. The results support the two catalytic sites acting sequentially, with negative cooperativity and product release being linked to motion of a catalytic site loop contributed by the neighboring subunit.

Can glucose be monitored accurately at the site of subcutaneous insulin delivery?

Because insulin promotes glucose uptake into adipocytes, it has been assumed that during measurement of glucose at the site of insulin delivery, the local glucose level would be much lower than systemic glucose. However, recent investigations challenge this notion. What explanations could account for a reduced local effect of insulin in the subcutaneous space? One explanation is that, in humans, the effect of insulin on adipocytes appears to be small. Another is that insulin monomers and dimers (from hexamer disassociation) might be absorbed into the circulation before they can increase glucose uptake locally. In addition, negative cooperativity of insulin action (a lower than expected effect of very high insulin concentrations)may play a contributing role. Other factors to be considered include dilution of interstitial fluid by the insulin vehicle and the possibility that some of the local decline in glucose might be due to the systemic effect of insulin. With regard to future research, redundant sensing units might be able to quantify the effects of proximity, leading to a compensatory algorithm. In summary, when measured at the site of insulin delivery, the decline in subcutaneous glucose level appears to be minimal, though the literature base is not large. Findings thus far support (1) the development of integrated devices that monitor glucose and deliver insulin and (2) the use of such devices to investigate the relationship between subcutaneous delivery of insulin and its local effects on glucose. A reduction in the number of percutaneous devices needed to manage diabetes would be welcome.

Single-Molecule Measurements to Investigate the Negative Cooperativity in Na+/K+ -ATPase

The Na+/K+-ATPase, a cell membrane ion motive ATPase, uses energy from the hydrolysis of ATP to move Na+ out of and K+ into cells, thus maintaining the membrane resting potential and cellular volume. To investigate how this pump functions, we isolated ATPase from duck supraorbital salt glands and labeled it with Cy3-maleimide (Cy3-ATPase). In bulk experiments, we found that the fluorescence of Cy3-ATPase decreases in the presence of ATP (Biochim Biophys Acta 2009; 1794:1549-1557). The kinetics of this ATP-induced fluorescence decrease exhibited negative cooperativity and could be explained in terms of protein aggregation. To further explore the phenomenon of negative cooperativity on the level of individual monomers, we used single-molecule total internal reflection fluorescence (SM-TIRF) microscopy. Protein monomers were solubilized and reconstituted into lipid vesicles to investigate the effect of varying ATP concentration on the fluorescence.Data from SM-TIRF experiments, analyzed using a hidden Markov model (HMM), suggest that the Cy3-ATPase exists in dynamic equilibrium between a high fluorescence state (unquenched) and a low fluorescence state (partially quenched). These kinetics are characterized by either rapid or slow transitions between these states. Two subpopulations are observed, one where the transitions between the states occur rapidly and the other where the kinetics are slower. Preliminary analysis of the data suggests that ATP shifts the population distribution from those exhibiting rapid transitions to those exhibiting slow transitions. Here, we report on the analysis of these effects and the implications of the above observations on the working of the pump.

Catenanes from catenanes: quantitative assessment of cooperativity in dynamic combinatorial catenation

A series of dynamic combinatorial [2] and [3]catenanes have been prepared. Formation of the [3]catenanes occurs with positive or negative cooperativity, depending on the cyclodextrin homologue. Systems level analysis allows cooperativity to be quantified and MD simulations reveal that cooperativity derives from the extents to which hydrophobic surface area is exposed to the aqueous surroundings.

Topological isomerism in a chiral handcuff catenane

The self-assembly of two topological isomers of a handcuff catenane has been achieved by utilizing the template-directed synthesis between the π-electron-rich bis-1,5-dioxynaphtho[50]crown-14 and the precursors to two fused π-electron-deficient cyclobis(paraquat-p-phenylene) cyclophanes. Characterization of the product using 1H NMR spectroscopy and single-crystal X-ray diffraction, with supporting density functional theory (DFT) calculations, suggests that the 1,5-dioxynaphthalene units in the major topological isomer align themselves with the same relative orientations inside the cyclophanes on account of restrictions imposed by the lengths of the polyether loops. The DFT calculations also reveal that the energies of the two topological isomers are similar to each other, supporting the experimental observation that both isomers can be isolated as a mixture from a one-pot reaction. The two isomers – designated as the meta–meta and ortho–ortho isomers with different topologies that are not interconvertible – only differ in the manner in which the polyether loops wind their way around the central 1,2,4,5-tetrasubstituted benzenoid ring in the ditopic host. X-Ray crystallography proves that by far the major topological isomer in the solid state is the meta–meta one. 1H NMR spectroscopy confirms that it is also the major isomer in solution, whilst also revealing the presence of a minor isomer, which is assumed, for the time-being, to have the ortho–ortho topology. The free fused ditopic host has been obtained using a protocol similar to that employed in the template-directed synthesis of the handcuff catenane, except that the crown ether is replaced with an acyclic template which can be removed post-synthesis. The results of isothermal titration calorimetry studies shed some light on the mechanism of binding of π-electron-rich guests: they lead us to believe that when two electron-rich guests bind to the ditopic host, they do so with allosteric negative cooperativity.

A hybrid bis(amino-styryl) substituted Bodipy dye and its conjugate diacid: synthesis, structure, spectroscopy and quantum chemical calculations

A new type of fluorescent pH indicator has been developed whereby two dissimilar amino-styryl units are attached to a boron dipyrromethene (Bodipy) dye. The photophysical properties of this hybrid dye, and its simpler counterparts bearing only a single amino-styryl residue, depend on the polarity of the surrounding medium. Of the two terminal amines, DFT (B3LYP/6-31G**) calculations and spectroscopic measurements support the notion that julolidine is oxidised and protonated under milder conditions than is N,N-dimethylaniline. For the hybrid dye, similar DFT calculations carried out for the mono-protonated analogues indicate that the julolidine residue is the stronger base while the resultant conjugate acid is the weaker one. Absorption and fluorescence spectroscopic titrations show that protonation of the hybrid dye occurs in two well-resolved steps, whereby addition of the first proton introduces a thermodynamic barrier for entry of the second. In the hybrid dye, the pKA values for the respective conjugate acids differ markedly from those derived for the mono-amino-styryl dyes and display negative co-operativity. Effectively, this means that electronic interactions running along the molecular backbone make it more difficult, relative to the individual dyes, to protonate both amino sites. As such, this dye operates as a probe over an unusually wide pH range.

Production, Partial Purification and Kinetic Characterization of Dextransucrase from Leuconostoc mesenteroides in Relation to Dextran Problem in Sugarcane

Dextransucrase was produced from Leuconostoc mesenteroides strain MTCC 107. Maximum enzyme production was observed after 60 h of incubation, with drop in pH from 7.0 at 0 h to 5.5 at 60 h. The crude enzyme was subjected to ammonium sulphate fractionation and DEAE-cellulose anion-exchange chromatography. Three isoforms of dextransucrase viz. DS-I, DS-II and DS-III, each with optimum pH 5.5 and optimum temperature 45 °C were identified. Isoforms DS-I and DS-III were found to be thermostable between 10 and 60 °C while isoform DS-II was thermostable between 10 and 50 °C. The Km values (for sucrose as substrate) of the isoforms DS-I, DS-II and DS-III at their optimum pH and temperature were 206.34, 492.45 and 108.00 mM, respectively. Corresponding Vmax values were 428.08, 505.58 and 410.85 μmol reducing sugars produced/min/mg protein, respectively. The pK a values of ionizing groups obtained by using Dixon and Marangoni methods suggested the participation of Asp/Glu residues and His residues in both substrate binding and catalysis. Lineweaver Burk plots obtained by varying concentrations of dextran (as substrate) at fixed sucrose concentration were found to be biphasic for all the three dextransucrase isoforms, suggesting negative cooperativity. Both the transferase and sucrase activities of the dextransucrase isoforms were markedly inhibited by sodium lauryl sulphate, barium chloride, EDTA, potassium chloride, manganese chloride and sodium metasilicate. These metal salts/chemicals when exogenously added to sugarcane juice effectively reduced the dextran formation during storage.

CYP2E1 hydroxylation of aniline involves negative cooperativity

CYP2E1 plays a role in the metabolic activation and elimination of aniline, yet there are conflicting reports on its mechanism of action, and hence relevance, in aniline metabolism. Based on our work with similar compounds, we hypothesized that aniline binds two CYP2E1 sites during metabolism resulting in cooperative reaction kinetics and tested this hypothesis through rigorous in vitro studies. The kinetic profile for recombinant CYP2E1 demonstrated significant negative cooperativity based on a fit of data to the Hill equation (n=0.56). Mechanistically, the data were best explained through a two-binding site cooperative model in which aniline binds with high affinity (Ks=30μM) followed by a second weaker binding event (Kss=1100uM) resulting in a threefold increase in the oxidation rate. Binding sites for aniline were confirmed by inhibition studies with 4-methylpyrazole. Inhibitor phenotyping experiments with human liver microsomes validated the central role for CYP2E1 in aniline hydroxylation and indicated minor roles for CYP2A6 and CYP2C9. Importantly, inhibition of minor metabolic pathways resulted in a kinetic profile for microsomal CYP2E1 that replicated the preferred mechanism and parameters observed with the recombinant enzyme. Scaled modeling of in vitro CYP2E1 metabolism of aniline to in vivo clearance, especially at low aniline levels, led to significant deviations from the traditional model based on non-cooperative, Michaelis–Menten kinetics. These findings provide a critical mechanistic perspective on the potential importance of CYP2E1 in the metabolic activation and elimination of aniline as well as the first experimental evidence of a negatively cooperative metabolic reaction catalyzed by CYP2E1.

Cooperativity of a few quantum emitters in a single-mode cavity

We theoretically investigate the emission properties of a single-mode cavity coupled to a mesoscopic number of incoherently pumped quantum emitters. We propose an operational measure for the medium cooperativity, valid both in the bad- and in the good-cavity regimes. We show that the opposite regimes of subradiance and super-radiance correspond to negative and positive cooperativity, respectively. The lasing regime is shown to be characterized by non-negative cooperativity. In the bad-cavity regime we show that the cooperativity defines the transitions from subradiance to super-radiance. In the good-cavity regime it helps to define the lasing threshold, also providing distinguishable signatures indicating the lasing regime. Increasing the quality factor of the cavity mode induces a crossover from the solely super-radiant to the lasing regime. Furthermore, we verify that lasing is manifested in a wide range of positive cooperative behavior, showing that stimulated emission and super-radiance can coexist. The robustness of the cooperativity is studied with respect to experimental imperfections, such as inhomogeneous broadening and pure dephasing.

Multisteric Regulation by Structural Disorder in Modular Signaling Proteins: An Extension of the Concept of Allostery

Allostery is a classical regulatory mechanism of proteins in which a signal at 'another site' modifies the activity/function of a protein. In fact, with the recognition of the generality of the structural disorder of proteins and the landscape theory of protein structure, a 'new view' of allostery started to emerge, in which emphasis is placed on ligand-induced shifts in the conformational ensemble of the protein. The ensuing changes in ligand binding/catalytic activity might stem from coupled folding transitions of distinct binding sites or remodeling of the conformational landscape to entropically favor a particular downstream binding/catalytic event. The ensuing sigmoidal binding isotherm cannot be described by a simple saturation; rather, it shows signs of cooperation between ligands. If binding of one ligand weakens that of the others, one can also speak about negative cooperativity. To elucidate the underlying mechanistic changes, two models have been suggested, which, even today, form the basis of our textbook wisdom of this phenomenon.

Controlling intramolecular hydrogen transfer in a porphycene molecule with single atoms or molecules located nearby

Although the local environment of a molecule can play an important role in its chemistry, rarely has it been examined experimentally at the level of individual molecules. Here we report the precise control of intramolecular hydrogen-transfer (tautomerization) reactions in single molecules using scanning tunnelling microscopy. By placing, with atomic precision, a copper adatom close to a porphycene molecule, we found that the tautomerization rates could be tuned up and down in a controlled fashion, surprisingly also at rather large separations. Furthermore, we extended our study to molecular assemblies in which even the arrangement of the pyrrolic hydrogen atoms in the neighbouring molecule influences the tautomerization reaction in a given porphycene, with positive and negative cooperativity effects. Our results highlight the importance of controlling the environment of molecules with atomic precision and demonstrate the potential to regulate processes that occur in a single molecule.

Avidity and positive allosteric modulation/cooperativity act hand in hand to increase the residence time of bivalent receptor ligands

Bivalent ligands bear two target-binding pharmacophores. Their simultaneous binding increases their affinity (avidity) and residence time. They become 'bitopic' when the binding sites at the target permit the pharmacophores the exert allosteric modulation of each other's affinity and/or activity. Present simulations reveal that positive cooperativity exacerbates these phenomena, whereas negative cooperativity curtails them, irrespective of whether the association or dissociation rates of the individual pharmacophores are affected. Positive cooperativity delays the attainment of equilibrium binding, yielding 'hemi-equilibrium' conditions and only apparent affinity constants under usual experimental conditions. Monovalent ligands that bind to one of the target sites decrease the bitopic ligand's residence time concentration-wise; their potency depends on their association rate and thereon acting cooperativity rather than on affinity. This stems from the repetitive, very fast reformation of fully bound bitopic ligand-target complexes by rebinding of freshly dissociated pharmacophores. These studies deal with kinetic binding properties (of increasing interest in pharmacology) of bitopic ligands (a promising avenue in medicinal chemistry).

Symmetric GroEL:GroES 2 complexes are the protein-folding functional form of the chaperonin nanomachine

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:GroES2 "football" particles regardless of the presence or absence of substrate protein (SP). In the absence of SP, these symmetric particles revert to asymmetric GroEL:GroES1 "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.