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Description, Altered ubiquitin conjugation is a mechanism by which mammalian cells can respond to fluctuating pCO2., The identification of CO2-binding proteins is crucial to understanding CO2-regulated molecular processes. CO2 can form a reversible posttranslational modification through carbamylation of neutral N-terminal α-amino or lysine ε-amino groups. We have previously developed triethyloxonium (TEO) ion as a chemical proteomics tool for covalent trapping of carbamates, and here, we deploy TEO to identify ubiquitin as a mammalian CO2-binding protein. We use 13C-NMR spectroscopy to demonstrate that CO2 forms carbamates on the ubiquitin N terminus and ε-amino groups of lysines 6, 33, 48, and 63. We demonstrate that biologically relevant pCO2 levels reduce ubiquitin conjugation at lysine-48 and down-regulate ubiquitin-dependent NF-κB pathway activation. Our results show that ubiquitin is a CO2-binding protein and demonstrates carbamylation as a viable mechanism by which mammalian cells can respond to fluctuating pCO2.

International audience; Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the cornerstone of atmospheric CO2 fixation by the biosphere. It catalyzes the addition of CO2 onto enolized ribulose 1,5-bisphosphate (RuBP), producing 3-phosphoglycerate which is then converted to sugars. The major problem of this reaction is competitive O-2 addition, which forms a phosphorylated product (2-phosphoglycolate) that must be recycled by a series of biochemical reactions (photorespiratory metabolism). However, the way the enzyme activates O-2 is still unknown. Here, we used isotope effects (with H-2, Mg-25, and O-18) to monitor O-2 activation and assess the influence of outer sphere atoms, in two Rubisco forms of contrasted O-2/CO2 selectivity. Neither the Rubisco form nor the use of solvent D2O and deuterated RuBP changed the O-16/O-18 isotope effect of O-2 addition, in clear contrast with the C-12/C-13 isotope effect of CO2 addition. Furthermore, substitution of light magnesium (Mg-24) by heavy, nuclear magnetic Mg-25 had no effect on O-2 addition. Therefore, outer sphere protons have no influence on the reaction and direct radical chemistry (intersystem crossing with triplet O-2) does not seem to be involved in O-2 activation. Computations indicate that the reduction potential of enolized RuBP (near 0.49 V) is compatible with superoxide (O-2(center dot-)) production, must be insensitive to deuteration, and yields a predicted O-16/O-18 isotope effect and energy barrier close to observed values. Overall, O-2 undergoes single electron transfer to form short-lived superoxide, which then recombines to form a peroxide intermediate.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the most widespread carboxylating enzyme in autotrophic organisms. Its kinetic and structural properties have been intensively studied for more than half a century. Yet important aspects of the catalytic mechanism remain poorly understood, especially the oxygenase reaction. Because of its relatively modest turnover rate (a few catalytic events per second) and the competitive inhibition by oxygen, Rubisco is often viewed as an inefficient catalyst for CO2 fixation. Considerable efforts have been devoted to improving its catalytic efficiency, so far without success. In this review, we re-examine Rubisco's catalytic performance by comparison with other chemically related enzymes. We find that Rubisco is not especially slow. Furthermore, considering both the nature and the complexity of the chemical reaction, its kinetic properties are unremarkable. Although not unique to Rubisco, oxygenation is not systematically observed in enolate and enamine forming enzymes and cannot be considered as an inevitable consequence of the mechanism. It is more likely the result of a compromise between chemical and metabolic imperatives. We argue that a better description of Rubisco mechanism is still required to better understand the link between CO2 and O2 reactivity and the rationale of Rubisco diversification and evolution.

Allosteric signaling in biological molecules, which may be viewed as specific action at a distance due to localized perturbation upon binding of ligands or changes in environmental cues, is pervasive in biology. Phenomenological MWC and KNF models galvanized research for over five decades, and these models continue to be the basis for describing the allostery in an array of systems. However, understanding allosteric signaling and the associated dynamics between distinct allosteric states is challenging. In this review, we first describe symmetry and rigidity as essential requirements for allosteric proteins. The general features, with MWC and KNF as two extreme scenarios, emerge when allosteric signaling is viewed from an energy landscape perspective. To go beyond the general theories, we describe computational tools to predict the network of residues that carry allosteric signals. Methods to obtain molecular insights into the dynamics of allosteric transitions are briefly mentioned. The utility of the methods is illustrated by applications to systems ranging from monomeric proteins to multisubunit proteins. Finally, the role allostery plays in the functions of ATP-consuming molecular machines, bacterial chaperonin GroEL and molecular motors, is described. Although universal molecular principles governing allosteric signaling do not exist, we can draw the following general conclusions. (1) Multiple pathways connecting allosteric states are highly heterogeneous. (2) Allosteric signaling is exquisitely sensitive to the architecture of the system, which implies that the capacity for allostery is encoded in the structure itself. (3) The mechanical modes that connect distinct allosteric states are robust to sequence variations. (4) Extensive investigations of allostery in Hemoglobin and more recently GroEL, show that a network of salt-bridge rearrangements serves as allosteric switches., 93 pages, 20 figures

Molecular chaperones are ATP-consuming biological machines, which facilitate the folding of proteins and RNA molecules that are kinetically trapped in misfolded states for long times. Unassisted folding occurs by the kinetic partitioning mechanism according to which folding to the native state, with low probability as well as misfolding to one of the many metastable states, with high probability, occur rapidly on similar time scales. GroEL is an all-purpose stochastic machine that assists misfolded substrate proteins (SPs) to fold. The RNA chaperones (CYT-19) help the folding of ribozymes that readily misfold. GroEL does not interact with the folded proteins but CYT-19 disrupts both the folded and misfolded ribozymes. Despite this major difference, the Iterative Annealing Mechanism (IAM) quantitatively explains all the available experimental data for assisted folding of proteins and ribozymes. Driven by ATP binding and hydrolysis and GroES binding, GroEL undergoes a catalytic cycle during which it samples three allosteric states, referred to as T (apo), R (ATP bound), and R'' (ADP bound). In accord with the IAM predictions, analyses of the experimental data shows that the efficiency of the GroEL-GroES machinery and mutants is determined by the resetting rate $k_{R''\rightarrow T}$, which is largest for the wild type GroEL. Generalized IAM accurately predicts the folding kinetics of Tetrahymena ribozyme and its variants. Chaperones maximize the product of the folding rate and the steady state native state fold by driving the substrates out of equilibrium. Neither the absolute yield nor the folding rate is optimized., Comment: 39 pages, 11 figures

Molecular chaperones facilitate the folding of proteins and RNA in vivo. Under physiological conditions, the in vitro folding of Tetrahymena ribozyme by the RNA chaperone CYT-19 behaves paradoxically; increasing the chaperone concentration reduces the yield of native ribozymes. In contrast, the protein chaperone GroEL works as expected; the yield of the native substrate increases with chaperone concentration. The discrepant chaperone-assisted ribozyme folding thus contradicts the expectation that it operates as an efficient annealing machine. To resolve this paradox, we propose a minimal stochastic model based on the Iterative Annealing Mechanism (IAM) that offers a unified description of chaperone-mediated folding of both proteins and RNA. Our theory provides a general relation that quantitatively predicts how the yield of native states depends on chaperone concentration. Although the absolute yield of native states decreases in the Tetrahymena ribozyme, the product of the folding rate and the steady-state native yield increases in both cases. By using energy from ATP hydrolysis, both CYT-19 and GroEL drive their substrate concentrations far out of equilibrium, thus maximizing the native yield in a short time. This also holds when the substrate concentration exceeds that of GroEL. Our findings satisfy the expectation that proteins and RNA be folded by chaperones on biologically relevant time scales, even if the final yield is lower than what equilibrium thermodynamics would dictate. The theory predicts that the quantity of chaperones in vivo has evolved to optimize native state production of the folded states of RNA and proteins in a given time.

Single-particle electron cryo-microscopy (cryo-EM) is an emerging tool for resolving structures of conformationally heterogeneous particles; however, each structure is derived from an average of many particles with presumed identical conformations. We used a 3.5-A cryo-EM reconstruction with imposed D7 symmetry to further analyze structural heterogeneity among chemically identical subunits in each GroEL oligomer. Focused classification of the 14 subunits in each oligomer revealed three dominant classes of subunit conformations. Each class resembled a distinct GroEL crystal structure in the Protein Data Bank. The conformational differences stem from the orientations of the apical domain. We mapped each conformation class to its subunit locations within each GroEL oligomer in our dataset. The spatial distributions of each conformation class differed among oligomers, and most oligomers contained 10–12 subunits of the three dominant conformation classes. Adjacent subunits were found to more likely assume the same conformation class, suggesting correlation among subunits in the oligomer. This study demonstrates the utility of cryo-EM in revealing structure dynamics within a single protein oligomer.

International audience; 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 new general tool, Fmoc-Ddae-OH, N-Fmoc-1-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-3-[2-(2-aminoethoxy)ethoxy]-propan-1-ol, a heterobifunctional 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 (Ebola virus 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 heterochiral 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.

In response to the binding of ATP, the two heptameric rings of the GroEL chaperonin protein interact with one another in a negatively cooperative manner. Owing to the helix dipole, the positively charged nitrogen of glycine 88 at the N-terminus of helix D binds to oxygen atoms on the β and γ phosphorus atoms of ATP. In apo-GroEL, the nucleotide-binding sites of different rings are connected to one another by the interaction of the ɛ-amino group of lysine 105 of one helix D across the twofold axis with the negatively charged carbonyl oxygen atom of alanine 109 at the C-terminus of the other helix D. Upon binding ATP, the K105–A109 salt bridge breaks and both helices move apart by approximately 3.5 Å en bloc toward the ATP. Upon hydrolysis of ATP, the helices return to their original position. The helices thus behave as pistons, their movement being driven by the binding and hydrolysis of ATP. This article is part of a discussion meeting issue ‘Allostery and molecular machines’.

The effects of potassium ion on the nested allostery of GroEL are due to increases in the affinity for nucleotide. Both positive allosteric transitions, TT-TR and TR-RR, occur at lower [ATP] as [K + ] is increased. Negative cooperativity in the double-ringed system is also due to an increase in the affinity of the trans ring for the product ADP as [K + ] is increased. Consequently, ( i ) rates of ATP hydrolysis are inversely proportional to [K + ] and ( ii ) the residence time of GroES bound to the cis ring is prolonged and the hemicycle time extended. Substrate protein suppresses negative cooperativity by decreasing the affinity of the trans ring for ADP, reducing the hemicycle time to a constant minimum. The trans ring thus serves as a variable timer. ATP added to the asymmetric GroEL-GroES resting-state complex lacking trans ring ADP is hydrolyzed in the newly formed cis ring with a presteady-state burst of ≈6 mol of Pi per mole of 14-mer. No burst is observed when the trans ring contains ADP. The amplitude and kinetics of ATP hydrolysis in the cis ring are independent of the presence or absence of encapsulated substrate protein and independent of K + at concentrations where there are profound effects on the linear steady-state rate. The hydrolysis of ATP by the cis ring constitutes a second, nonvariable timer of the chaperonin cycle.

Chaperonins cpn60 and cpn10 are essential proteins involved in cellular protein folding. Plant chloroplasts contain a unique version of the cpn10 co-chaperonin, cpn20, which consists of two homologous cpn10-like domains (N-cpn20 and C-cpn20) that are connected by a short linker region. Although cpn20 seems to function like other single domain cpn10 oligomers, the structure and specific functions of the domains are not understood. We mutated amino acids in the "mobile loop" regions of N-cpn20, C-cpn20 or both: a highly conserved glycine, which was shown to be important for flexibility of the mobile loop, and a leucine residue shown to be involved in binding of co-chaperonin to chaperonin. The mutant proteins were purified and their oligomeric structure validated by gel filtration, native gel electrophoresis, and circular dichroism. Functional assays of protein refolding and inhibition of GroEL ATPase both showed (i) mutation of the conserved glycine reduced the activity of cpn20, whether in N-cpn20 (G32A) or C-cpn20 (G130A). The same mutation in the bacterial cpn10 (GroES G24A) had no effect on activity. (ii) Mutations in the highly conserved leucine of N-cpn20 (L35A) and in the corresponding L27A of GroES resulted in inactive protein. (iii) In contrast, mutant L133A, in which the conserved leucine of C-cpn20 was altered, retained 55% activity. We conclude that the structure of cpn20 is much more sensitive to alterations in the mobile loop than is the structure of GroES. Moreover, only N-cpn20 is necessary for activity of cpn20. However, full and efficient functioning requires both domains.

We have used a bioinformatic approach to predict the natural substrate proteins for the Escherichia coli chaperonin GroEL based on two simple criteria. Natural substrate proteins should contain binding motifs similar in sequence to the mobile loop peptide of GroES that displaces the binding motif during the chaperonin cycle. Secondly, each substrate protein should contain multiple copies of the binding motif so that the chaperonin can perform “work” on the substrate protein. To validate these criteria, we have used a database of 252 proteins that have been experimentally shown to interact with the chaperonin machinery in vivo . More than 80% are identified by these criteria. The binding motifs of all 79 proteins in the database with a known three-dimensional structure are buried (

The ability of the GroEL chaperonin to unfold a protein trapped in a misfolded condition was detected and studied by hydrogen exchange. The GroEL-induced unfolding of its substrate protein is only partial, requires the complete chaperonin system, and is accomplished within the 13 seconds required for a single system turnover. The binding of nucleoside triphosphate provides the energy for a single unfolding event; multiple turnovers require adenosine triphosphate hydrolysis. The substrate protein is released on each turnover even if it has not yet refolded to the native state. These results suggest that GroEL helps partly folded but blocked proteins to fold by causing them first to partially unfold. The structure of GroEL seems well suited to generate the nonspecific mechanical stretching force required for forceful protein unfolding.

The interaction of the chaperonin GroEL 14 with its cochaperonin GroES 7 is dynamic, involving stable, asymmetric 1:1 complexes (GroES 7 ⋅GroEL 7 –GroEL 7 ) and transient, metastable symmetric 2:1 complexes [GroES 7 ⋅GroEL 7 –GroEL 7 ⋅GroES 7 ]. The transient formation of a 2:1 complex permits exchange of free GroES 7 for GroES 7 bound in the stable 1:1 complex. Electrophoresis in the presence of ADP was used to resolve free GroEL 14 from the GroES 7 –GroEL 14 complex. Titration of GroEL 14 with radiolabeled GroES 7 to molar ratios of 32:1 demonstrated a 1:1 limiting stoichiometry in a stable complex. No stable 2:1 complex was detected. Preincubation of the asymmetric GroES 7 ⋅GroEL 7 –GroEL 7 complex with excess unlabeled GroES 7 in the presence of ADP demonstrated GroES 7 exchange. The rates of GroES 7 exchange were proportional to the concentration of unlabeled free GroES 7 . This concentration dependence points to an associative mechanism in which exchange of GroES 7 occurs by way of a transient 2:1 complex and excludes a dissociative mechanism in which exchange occurs by way of free GroEL 14 . Exchange of radiolabeled ADP from 1:1 complexes was much slower than the exchange of GroES 7 . In agreement with recent structural studies, this indicates that conformational changes in GroEL 14 following the dissociation of GroES 7 must precede ADP release. These results explain how the GroEL 14 cavity can become reversibly accessible to proteins under in vivo conditions that favor 2:1 complexes.

Dethiobiotin synthetase (DTBS; E.C. 6.6.6.6), the penultimate enzyme in the biosynthesis of the essential vitamin biotin, is a new potential target for novel herbicides. Inhibitors were designed based on mechanistic and structural information. The in-vitro activities of these potential inhibitors versus the bacterial enzyme are reported here. Mimics of 7,8-diaminopelargonic acid (DAPA) or the DAPA carbamate reaction intermediate were substrates or partial substrates for the enzyme. Synergistic binding with ATP was noted with compounds which contained an amino functionality. NMR studies and X-ray structures confirmed that the inhibitors could be phosphorylated by the enzyme. Several series of potential inhibitors were designed to take advantage of this partial substrate activity by generating potentially more tightly bound phosphorylated inhibitors in situ. Structure-activity relationships for these series based on both substrate and inhibitory activity are described herein. An X-ray structure for one of these inhibitors is also discussed. Although considerable potential for inhibitors of this type was demonstrated, none of the compounds reported showed sufficient herbicidal activity to be a commercial proposition.

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.

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-GroES1 “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-GroES2 football complex that is, consequently, the predominant species. A FRET-based analysis confirms that asymmetric GroEL-GroES1 bullets predominate in the absence of SP, whereas symmetric GroEL-GroES2 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.

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 cross-links 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.

Chaperonins prevent the aggregation of partially folded or misfolded forms of a protein and, thus, keep it competent for productive folding. It was suggested that GroEL, the chaperonin of Escherichia coli, exerts this function 1 unfolding such intermediates, presumably in a catalytic fashion. We investigated the kinetic mechanism of GroEL-induced protein unfolding by using a reduced and carbamidomethylated variant of RNase T1, RCAM-T1, as a substrate. RCAM-T1 cannot fold to completion, because the two disulfide bonds are missing, and it is, thus, a good model for long-lived folding intermediates. RCAM-T1 unfolds when GroEL is added, but GroEL does not change the microscopic rate constant of unfolding, ruling out that it catalyzes unfolding. GroEL unfolds RCAM-T1 because it binds with high affinity to the unfolded form of the protein and thereby shifts the overall equilibrium toward the unfolded state. GroEL can unfold a partially folded or misfolded intermediate by this thermodynamic coupling mechanism when the Gibbs free energy of the binding to GroEL is larger than the conformational stability of the intermediate and when the rate of its unfolding is high.

The stuctures of the co-chaperonin GroES and of the GroEL•ATPγS complex raise a host of tantalizing questions and whet the appetite for even more challenging structures, the various GroEL•nucleotide•GroES complexes which facilitate folding.

The chaperonin proteins, GroEL14 and GroES7, inhibit protein aggregation and assist in protein folding in a potassium/ATP-dependent manner. In vitro, assays for chaperonin activity typically involve adding a denatured substrate protein to the chaperonins and measuring the appearance of correctly folded substrate protein. The influence of denaturant is generally ignored. Low concentrations of guanidinium chloride (

The particular structural arrangement of chaperonins probably contributes to their ability to assist in the folding of proteins. The interaction of the oligomeric bacterial chaperonin GroEL and its cochaperonin, GroES, in the presence of adenosine diphosphate (ADP) forms an asymmetric complex. However, in the presence of adenosine triphosphate (ATP) or its nonhydrolyzable analogs, symmetric complexes were found by electron microscopy and image analysis. The existence of symmetric chaperonin complexes is not predicted by current models of the functional cycle for GroE-mediated protein folding. Because complete folding of a nonnative substrate protein in the presence of GroEL and GroES only occurs in the presence of ATP, but not with ADP, the symmetric chaperonin complexes formed during the GroE cycle are proposed to be functionally significant.

Earlier structural and functional studies of ribulose-1,5-bisphosphate carboxylase/oxygenase imply that K334 facilitates the addition of gaseous substrate to the 2,3-enediol(ate) derived from ribulose 1,5-bisphosphate. Crystallographic analysis of the activated spinach enzyme [Knight et al. (1990) J. Mol. Biol. 215, 113-160] shows that the lysyl side chain is appropriately positioned to stabilize the transition state for the addition of CO2 to the enediol(ate). Furthermore, despite total impairment of carboxylase and oxygenase activities, site-directed mutants of the Rhodospirillum rubrum enzyme with replacements for lysine K334 (formerly designated K329) retain the capacity to enolize ribulose bisphosphate, demonstrating that the primary catalytic lesion lies beyond this initial step [Soper et al. (1988) Protein Eng. 2, 39-44; Hartman & Lee (1989) J. Biol. Chem. 264, 11784-11789]. We now show that the K334C mutant is also competent in the latter stages of catalysis, whereby 2'-carboxy-3-keto-D-arabinitol 1,5-bisphosphate, the six-carbon intermediate of the carboxylation pathway, is correctly processed to 3-phosphoglycerate. Thus, the impairment of the mutant in overall catalysis can be attributed to preferential disruption of the reaction of CO2 or O2 with the enzyme-bound enediol(ate). Chemical rescue of the K334C mutant by aminoethylation and aminopropylation shows that this disruption reflects, at least in part, a failure to adequately stabilize the relevant transition state. With several simplifying assumptions, the CO2/O2 specificity factor tau can be reduced to the ratio of the fundamental second-order rate constants for the interaction of the gaseous substrates with the enzyme-bound 2,3-enediol(ate) of ribulose bisphosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Dimer-monomer dissociation of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum was investigated using hydrostatic pressure in the range 1-2 kbar to promote dissociation. Intrinsic fluorescence emission and polarization, along with the polarization of the fluorescence of single-labeled AEDANS conjugates, were used to follow the dissociation. Full reversibility after dissociation was observed to depend on the presence of small ligands: glycerol, Mg2+, and NaHCO3, the last two being required to activate the enzyme. The free energy of association at 15 degrees C, -12.9 kcal mol-1, was made up of a positive change in enthalpy on association of 6.0 kcal mol-1 and an entropic contribution (T delta S) of 18.9 kcal mol-1; thus the monomer association is entropy driven. No dissociation of the quaternary complex formed by the dimer, 2-carboxy-D-arabinitol 1,5-diphosphate (CADP), Mg2+, and NaHCO3 was observed at pressures up to 2.0 kbar; the magnitude of stabilization by the inhibitor binding was estimated as 2.3 kcal mol-1. Pressurization in the presence of bis-ANS results in a time-dependent increase in fluorophore emission, indicating changes in monomer conformation with exposure of hydrophobic surfaces upon dissociation. Reactivity against the fluorescent probe 1,5-I-AEDANS was also used as a conformational probe: HPLC of a trypsin digest of rubisco labeled at atmospheric pressure revealed a single fluorescent peptide, whereas more extensive labeling was observed when the reaction was carried out at 2.0 kbar, indicative of exposure of internal cysteines.(ABSTRACT TRUNCATED AT 250 WORDS)

Chaperonin-facilitated folding of proteins involves two partial reactions. The first partial reaction, the formation of stable binary complexes between chaperonin-60 and non-native states of the target protein, is common to the chaperonin-facilitated folding of all target proteins investigated to date. The structural basis for this interaction is not presently understood. The second partial reaction, the dissociation of the target protein in a form committed to the native state, appears to proceed by a variety of mechanisms, dependent upon the nature of the target protein in question. Those target proteins (e.g. rubisco, rhodanese, citrate synthase) which require the presence of chaperonin-10, also appear to require the hydrolysis of ATP to bring about the dissociation of the target protein from chaperonin-60. With one exception (pre-β-lactam ase) those target proteins which do not require the presence of chaperonin-10 to be released from chaperonin-60, also do not require the hydrolysis of ATP, since non-hydrolysable analogues of ATP support the release of the target protein in a state committed to the native state. The question of whether or not chaperonin-facilitated folding constitutes a catalysed event is addressed.

Chaperonins are thought to participate in the process of protein folding in bacteria and in eukaryotic mitochondria and chloroplasts. While some chaperonins are relatively well characterized, the structures of the mammalian chaperonins are unknown. We have expressed a mammalian mitochondrial chaperonin 60 in Escherichia coli and purified the recombinant protein to homogeneity. Structural and biochemical analyses of this protein establish a single toroidal structure of seven subunits, in contrast to the homologous bacterial, fungal, and plant chaperonin 60s, which have double toroidal structures comprising two layers of seven identical subjects each. The recombinant mammalian chaperonin 60, together with the mammalian chaperonin 10 (but not with bacterial chaperonin 10), facilitates the formation of catalytically active ribulose-bisphosphate carboxylase from an unfolded state in the presence of K+ and MgATP. Analysis of the partial reactions involved in this in vitro reconstitution reveals that the single toroid of chaperonin 60 can form stable complexes with both unfolded or partially folded [35S]ribulose-bisphosphate carboxylase and mitochondrial (but not bacterial) chaperonin 10 in the presence of MgATP. We conclude that the minimal functional unit of chaperonin 60 is a single hepatmeric toroid.

GroEL is a bacterial chaperone protein that assembles into a homotetradecameric complex exhibiting D(7) symmetry and utilizes the co-chaperone protein GroES and ATP hydrolysis to assist in the proper folding of a variety of cytosolic proteins. GroEL utilizes two metal cofactors, Mg(2+) and K(+), to bind and hydrolyze ATP. A K(+)-binding site has been proposed to be located next to the nucleotide-binding site, but the available structural data do not firmly support this conclusion. Moreover, more than one functionally significant K(+)-binding site may exist within GroEL. Because K(+) has important and complex effects on GroEL activity and is involved in both positive (intra-ring) and negative (inter-ring) cooperativity for ATP hydrolysis, it is important to determine the exact location of these cation-binding site(s) within GroEL. In this study, the K(+) mimetic Tl(+) was incorporated into GroEL crystals, a moderately redundant 3.94 A resolution X-ray diffraction data set was collected from a single crystal and the strong anomalous scattering signal from the thallium ion was used to identify monovalent cation-binding sites. The results confirmed the previously proposed placement of K(+) next to the nucleotide-binding site and also identified additional binding sites that may be important for GroEL function and cooperativity. These findings also demonstrate the general usefulness of Tl(+) for the identification of monovalent cation-binding sites in protein crystal structures, even when the quality and resolution of the diffraction data are relatively low.

In a study of the timing mechanism of the chaperonin nanomachine we show that the hemicycle time (HCT) is determined by the mean residence time (MRT) of GroES on the cis ring of GroEL. In turn, this is governed by allosteric interactions within the trans ring of GroEL. Ligands that enhance the R (relaxed) state (residual ADP, the product of the previous hemicycle, and K + ) extend the MRT and the HCT, whereas ligands that enhance the T (taut) state (unfolded substrate protein, SP) decrease the MRT and the HCT. In the absence of SP, the chaperonin machine idles in the resting state, but in the presence of SP it operates close to the speed limit, set by the rate of ATP hydrolysis by the cis ring. Thus, the conformational states of the trans ring largely control the speed of the complete chaperonin cycle.

Both the chaperonin- and MgATP-dependent reconstitution of unfolded ribulosebisphosphate carboxylase (Rubisco) and the uncoupled ATPase activity of chaperonin 60 (groEL) require ionic potassium. The spontaneous, chaperonin-independent reconstitution of Rubisco, observed at 15 but not at 25 degrees C, requires no K+ and is actually inhibited by chaperonin 60, with which the unfolded or partly folded Rubisco forms a stable binary complex. The chaperonin-dependent reconstitution of Rubisco involves the formation of a complex between chaperonin 60 and chaperonin 10 (groES). Formation of this complex almost completely inhibits the uncoupled ATPase activity of chaperonin 60. Furthermore, although the formation of the chaperonin 60-chaperonin 10 complex requires the presence of MgATP, hydrolysis of ATP may not be required, since complex formation occurs in the absence of K+. The interaction of chaperonin 60 with unfolded or partly folded Rubisco does not require MgATP, K+, or chaperonin 10. However, discharge of the complex of chaperonin 60-Rubisco, which leads to the formation of active Rubisco dimers, requires chaperonin 10 and a coupled, K(+)-dependent hydrolysis of ATP. We propose that a role of chaperonin 10 is to couple the K(+)-dependent hydrolysis of ATP to the release of the folded monomers of the target protein from chaperonin 60.

The chaperonin GroEL-GroES, a machine which helps some proteins to fold, cycles through a number of allosteric states, the $T$ state, with high affinity for substrate proteins (SPs), the ATP-bound $R$ state, and the $R^{\prime\prime}$ ($GroEL-ADP-GroES$) complex. Structures are known for each of these states. Here, we use a self-organized polymer (SOP) model for the GroEL allosteric states and a general structure-based technique to simulate the dynamics of allosteric transitions in two subunits of GroEL and the heptamer. The $T \to R$ transition, in which the apical domains undergo counter-clockwise motion, is mediated by a multiple salt-bridge switch mechanism, in which a series of salt-bridges break and form. The initial event in the $R \to R^{\prime\prime}$ transition, during which GroEL rotates clockwise, involves a spectacular outside-in movement of helices K and L that results in K80-D359 salt-bridge formation. In both the transitions there is considerable heterogeneity in the transition pathways. The transition state ensembles (TSEs) connecting the $T$, $R$, and $R^{\prime\prime}$ states are broad with the the TSE for the $T \to R$ transition being more plastic than the $R\to R^{\prime\prime}$ TSE. The results suggest that GroEL functions as a force-transmitting device in which forces of about (5-30) pN may act on the SP during the reaction cycle., 32 pages, 10 figures (Longer version than the one published)

▪ Abstract Molecular chaperones are required to assist folding of a subset of proteins in Escherichia coli. We describe a conceptual framework for understanding how the GroEL-GroES system assists misfolded proteins to reach their native states. The architecture of GroEL consists of double toroids stacked back-to-back. However, most of the fundamentals of the GroEL action can be described in terms of the single ring. A key idea in our framework is that, with coordinated ATP hydrolysis and GroES binding, GroEL participates actively by repeatedly unfolding the substrate protein (SP), provided that it is trapped in one of the misfolded states. We conjecture that the unfolding of SP becomes possible because a stretching force is transmitted to the SP when the GroEL particle undergoes allosteric transitions. Force-induced unfolding of the SP puts it on a higher free-energy point in the multidimensional energy landscape from which the SP can either reach the native conformation with some probability or be trapped in one of the competing basins of attraction (i.e., the SP undergoes kinetic partitioning). The model shows, in a natural way, that the time scales in the dynamics of the allosteric transitions are intimately coupled to folding rates of the SP. Several scenarios for chaperonin-assisted folding emerge depending on the interplay of the time scales governing the cycle. Further refinement of this framework may be necessary because single molecule experiments indicate that there is a great dispersion in the time scales governing the dynamics of the chaperonin cycle.

The E. coli chaperonin proteins, GroEL and GroES, assist in folding newly synthesized proteins. GroES is necessary for GroEL-assisted folding under conditions where the substrate protein cannot spontaneously fold. On the basis of photolabelling of GroES with 8-azido-ATP, a role for nucleotide binding to GroES in chaperonin function was suggested [Martin, et al., Nature, 366 (1993) 279–2821]. We confirm the photolabelling of GroES with 8-azido-ATP. However, other proteins not known to contain nucleotide binding sites also become photolabeled suggesting that labeling is non-specific. Using rigorous physical methods, isothermal calorimetry and equilibrium binding, no interaction between GroES and nucleotides could be detected. We conclude that GroES has no nucleotide binding site.

The Escherichia coli chaperonin system, GroEL-GroES, facilitates folding of substrate proteins (SPs) that are otherwise destined to aggregate. The iterative annealing mechanism suggests that the allostery-driven GroEL transitions leading to changes in the microenvironment of the SP constitutes the annealing action of chaperonins. To describe the molecular basis for the changes in the nature of SP-GroEL interactions we use the crystal structures of GroEL (T state), GroEL-ATP (R state) and the GroEL-GroES-(ADP)(7) (R" state) complex to determine the residue-specific changes in the accessible surface area and the number of tertiary contacts as a result of the T-->R-->R" transitions. We find large changes in the accessible area in many residues in the apical domain, but relatively smaller changes are associated with residues in the equatorial domain. In the course of the T-->R transition the microenvironment of the SP changes which suggests that GroEL is an annealing machine even without GroES. This is reflected in the exposure of Glu386 which loses six contacts in the T-->R transition. We also evaluate the conservation of residues that participate in the various chaperonin functions. Multiple sequence alignments and chemical sequence entropy calculations reveal that, to a large extent, only the chemical identities and not the residues themselves important for the nominal functions (peptide binding, nucleotide binding, GroES and substrate protein release) are strongly conserved. Using chemical sequence entropy, which is computed by classifying aminoacids into four types (hydrophobic, polar, positively charged and negatively charged) we make several new predictions that are relevant for peptide binding and annealing function of GroEL. We identify a number of conserved peptide binding sites in the apical domain which coincide with those found in the 1.7 A crystal structure of 'mini-chaperone' complexed with the N-terminal tag. Correlated mutations in the HSP60 family, that might control allostery in GroEL, are also strongly conserved. Most importantly, we find that charged solvent-exposed residues in the T state (Lys 226, Glu 252 and Asp 253) are strongly conserved. This leads to the prediction that mutating these residues, that control the annealing function of the SP, can decrease the efficacy of the chaperonin function.

Unlike the GroEL homologs of eubacteria and mitochondria, oligomer preparations of the higher plant chloroplast chaperonin 60 (cpn60) consist of roughly equal amounts of two divergent subunits, alpha and beta. The functional significance of these isoforms, their structural organization into tetradecamers, and their interactions with the unique binary chloroplast chaperonin 10 (cpn10) have not been elucidated. Toward this goal, we have cloned the alpha and beta subunits of the ch-cpn60 of pea (Pisum sativum), expressed them individually in Escherichia coli, and subjected the purified monomers to in vitro reconstitution experiments. In the absence of other factors, neither subunit (alone or in combination) spontaneously assembles into a higher order structure. However, in the presence of MgATP, the beta subunits form tetradecamers in a cooperative reaction that is potentiated by cpn10. In contrast, alpha subunits only assemble in the presence of beta subunits. Although beta and alpha/beta 14-mers are indistinguishable by electron microscopy and can both assist protein folding, their specificities for cpn10 are entirely different. Similar to the authentic chloroplast protein, the reconstituted alpha/beta 14-mers are functionally compatible with bacterial, mitochondrial, and chloroplast cpn10. In contrast, the folding reaction mediated by the reconstituted beta 14-mers is only efficient with mitochondrial cpn10. The ability to reconstitute two types of functional oligomer in vitro provides a unique tool, which will allow us to investigate the mechanism of this unusual chaperonin system.

Publisher Summary The chapter discusses criteria for the assessment of the purity and quality of GroEL. The chapter discusses removing contaminating proteins from GroEL, tryptophan determination, and silver staining of sodium dodecyl sulfate-polyacrylamide gels. The method of tryptophan determination employs bovine serum albumin (containing two tryptophan residues per protomer) in place of free tryptophan. The preparations of GroEL used in these experiments were severely contaminated by an ensemble of unfolded proteins. Therefore, the way such a stable complex could be created in the presence of reagents (unfolded proteins) that induce its dissociation. In a similar vein, conclusions concerning the stoichiometry of protein binding to GroEL, the influence of unfolded proteins on the basal ATPase activity of GroEL, and other similar parameters are likely to be compromised, when the experiments are performed with preparations of GroEL already containing significant quantities of contaminating protein. GroEL binds to a wide variety of unfolded proteins with dissociation constants varying from micromolar to picomolar. GroEL contains no tryptophan residues. Thus, the presence of tryptophan in a preparation of GroEL can be taken as an indicator of contamination. Tryptophan is one of the rarer of the natural amino acids.

Publisher Summary The chapter presents a study on purification of mammalian mitochondrial chaperonin 60 through in vitro reconstitution of active oligomers. Because of the technical difficulties associated with the mammalian mt-cpn60, functional characterization of the mammalian mt-cpnl0 has largely been restricted to in vitro interactions with GroEL. In this heterologous test system, GroES and the mammalian mt-cpnl 0 are functionally interchangeable by a number of criteria, including the ability to assist GroEL in the facilitation of protein folding. The resounding conclusion from these experiments is that the fundamental mechanism of the GroE-related chaperonins has been highly conserved from bacteria to mitochondria. The chapter describes the purification of monomeric mammalian mt-cpn60 and its subsequent reassembly into functional oligomers using a general approach that has worked well with other GroEL homologs. The chapter describes the purification of monomeric mammalian mitochondrial chaperonin 60, reconstitution of oligomeric mitochondrial chaperonin 60, properties of in vitro -reconstituted particles and several related concepts.

We develop a heuristic model for chaperonin-facilitated protein folding, the iterative annealing mechanism, based on theoretical descriptions of "rugged" conformational free energy landscapes for protein folding, and on experimental evidence that (i) folding proceeds by a nucleation mechanism whereby correct and incorrect nucleation lead to fast and slow folding kinetics, respectively, and (ii) chaperonins optimize the rate and yield of protein folding by an active ATP-dependent process. The chaperonins GroEL and GroES catalyze the folding of ribulose bisphosphate carboxylase at a rate proportional to the GroEL concentration. Kinetically trapped folding-incompetent conformers of ribulose bisphosphate carboxylase are converted to the native state in a reaction involving multiple rounds of quantized ATP hydrolysis by GroEL. We propose that chaperonins optimize protein folding by an iterative annealing mechanism; they repeatedly bind kinetically trapped conformers, randomly disrupt their structure, and release them in less folded states, allowing substrate proteins multiple opportunities to find pathways leading to the most thermodynamically stable state. By this mechanism, chaperonins greatly expand the range of environmental conditions in which folding to the native state is possible. We suggest that the development of this device for optimizing protein folding was an early and significant evolutionary event.

In vitro the chaperonin proteins, GroEL and GroES, facilitate the folding of some other proteins under conditions where that process does not occur spontaneously. Using values drawn from a number of such in vitro studies, together with the known rates of in vivo protein synthesis by Escherichia coli and the known quantities of GroEL and GroES in E. coli, an assessment of the general role of these proteins in protein folding in vivo has been made. Three specific cases are examined, where compelling evidence points to the involvement of the chaperonins; the in vivo folding of the bacteriophage coat protein during the burst phase of phage morphogenesis and of Rubisco during chloroplast development and during expression of recombinant Rubisco in E. coli. In each case the maximum in vitro rates are nearly sufficient to account for the observed in vivo rates of formation of the native protein. However, in general, there appears to be sufficient GroEL and GroES to facilitate the folding of no more than 5% of all of the proteins within E. coli.

Dethiobiotin synthetase (DTBS) catalyzes the penultimate step in biotin biosynthesis, the formation of the ureido ring of dethiobiotin from (7R,8S)-7,8-diaminononanoic acid (7,8-diaminopelargonic acid, DAPA), CO2, and ATP. Solutions of DAPA at neutral pH readily formed a mixture of the N7- and N8-carbamates in the presence of CO2. However, four lines of evidence together indicated that only the N7-carbamate of DAPA was an intermediate in the reaction catalyzed by DTBS. (1) Addition of diazomethane to mixtures of DAPA and [14C]CO2 yielded a mixture of the N7- and N8-methyl carbamate esters, consistent with carbamate formation in free solution. In the presence of excess DTBS (over DAPA), the ratio of N7:N8-methyl carbamate esters recovered was roughly doubled, suggesting that the enzyme preferentially bound the N7-DAPA-carbamate. (2) Both N7- and N8-DAPA-carbamates were observed directly by 1H and 13C NMR in solutions containing DAPA and [13C]CO2. In the presence of excess DTBS (over DAPA) only one carbamate was observed, showing that carbamate binding to the enzyme was regiospecific. 13C NMR of mixtures containing enzyme, [7-15N]DAPA, and [13C]CO2 showed that the enzyme-bound carbamate was at N7 of DAPA. In addition, pulse-chase experiments showed that the binary complex of DTBS and N7-DAPA-carbamate became kinetically committed upon addition of MgATP. (3) The N7-DAPA-carbamate mimic, 3-(1-aminoethyl)nonanedioic acid, in which the carbamate nitrogen was replaced with a methylene group, cyclized to the corresponding lactam in the presence of DTBS and ATP; ADP and P(i) were also formed.(ABSTRACT TRUNCATED AT 250 WORDS)

The X-ray structure of GroEL puts future studies on a firm footing, but there's still much work to be done before chaperonin-assisted protein folding is understood.

The potassium-ion activation constant (Kact) for the ATPase activity of Escherichia coli chaperonin groEL is inversely dependent upon the ATP concentration over at least 3 orders of magnitude. The ATPase activity shows positively cooperative kinetics with respect to ATP and K+. Both the K0.5 for ATP and cooperativity (as measured by the Hill coefficient) decrease as the K+ concentration increases. Equilibrium binding studies under conditions where hydrolysis does not occur indicate that MgATP binds weakly to groEL in the absence of K+. In the absence of groES, the K(+)-dependent hydrolysis of ATP by groEL continues to completion. In the presence of groES, the time course for the hydrolysis of ATP by groEL becomes more complex. Three distinct kinetic phases can be discerned. Initially, both heptameric toroids turn over once at the same rate that they do in the absence of groES. This leads to the formation of an asymmetric binary complex, groEL14-MgADP7-groES7, in which 7 mol of ADP is trapped in a form that does not readily exchange with free ADP. In the second phase, the remaining seven sites (containing readily exchangeable ADP) turn over, or have the potential to turn over, at the same rate as they do in the absence of groES, so that the overall rate of hydrolysis is maximally 50%. These remaining sites of the asymmetric binary complex do not hydrolyze all of the available ATP. Instead, the second phase of hydrolysis gives way to a third, completely inhibited state, the onset of which is dependent upon the relative affinities of the remaining sites for MgATP and MgADP.(ABSTRACT TRUNCATED AT 250 WORDS)

Conformational states of ribulosebisphosphate carboxylase (Rubisco) from Rhodospirillum rubrum were examined by far-UV circular dichroism (CD), tryptophan fluorescence, and 1-anilino-naphthalenesulfonate (ANS) binding. At pH 2 and low ionic strength (I = 0.01), Rubisco adopts an unfolded, monomeric conformation (UA1 state) as judged by far-UV CD and tryptophan fluorescence. As with other acid-unfolded proteins [Goto, Y., Calciano, L. J., & Fink, A. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 573-577], an intermediate conformation (A1 state) is observed at pH 2 and high ionic strength. The A1 state has an alpha-helical content equivalent to 64% of that present in the native dimer (N2 state). However, fluorescence measurements indicate that the tertiary structure of the A1 state is largely disordered. A site-directed mutant, K168E, which exists as a stable monomer [Mural, R. J., Soper, T. S., Larimer, F. W., & Hartman, F. C. (1990) J. Biol. Chem. 265, 6501-6505] was used to characterize the "native" monomer (N1 state). The far-UV CD spectra of the N1 and N2 states are almost identical, indicating a similar secondary structure content. However, the tertiary structure of the N1 state is less ordered than that of the N2 state. Nevertheless, when appropriately complemented in vitro, K168E forms an active heterodimer. Upon neutralization of acid-denatured Rubisco or dilution of guanidine hydrochloride-denatured Rubisco, unstable folding intermediates (I1 state) are rapidly formed. At concentrations at or below the "critical aggregation concentration" (CAC), the I1 state reverts spontaneously but slowly to the native states with high yield (greater than 65%). The CAC is temperature-dependent. At concentrations above the CAC, the I1 and the A1 states undergo irreversible aggregation. The commitment to aggregation is rapid [ef. Goldberg, M. E., Rudolph, R., & Jaenicke, R. (1991) Biochemistry 30, 2790-2797] and proceeds until the concentration of folding intermediate(s) has fallen to the CAC. In the presence of a molar excess of chaperonin 60 oligomers, the I1 state forms a stable binary complex. No stable binary complex between chaperonin 60 and the N1 state could be detected. Formation of the chaperonin 60-I1 binary complex arrests the spontaneous folding process. The I1 state becomes resistant to interaction with chaperonin 60 with kinetics indistinguishable from those associated with the appearance of the native states. In vitro complementation analysis indicated that the product of the chaperonin-facilitated process is monomeric.(ABSTRACT TRUNCATED AT 400 WORDS)

Atomic force microscopy (AFM) is an elegant technique in which a roving microscopic needle tip provides images of the surfaces of objects beneath it. In a Perspective, Forbes and Lorimer discuss how AFM can be applied to the step-wise unfolding of membrane proteins such as bacteriorhodopsin ( Oesterhelt et al.). When the AFM tip becomes attached to points on the protein exposed on the membrane surface, the protein unfolds, helix by helix, as the tip is withdrawn.

Crystals of the hexadecameric form of ribulose-bisphosphate carboxylase used to solve the structure of the enzyme are composed of protein substantially crosslinked by a disulfide bond between pairs of large subunits. Conditions leading to the selective formation of dimers of the large subunits are described. The stability and specificity of the intra-dimeric crosslink was used to confirm that only one cysteine residue, Cys247 of neighboring large subunits, is involved in the bridge. The ability to generate this disulfide selectively, or alternatively replace the cysteine by site-directed mutagenesis, has led us to conclude that there is no effect of these changes on any of the critical kinetic parameters of the enzyme. The benign effect of the oxidation indicates that the crystal structures of the ribulose-bisphosphate carboxylase, particularly of the active site, are a true representation of the native enzyme.