Publications

Characterization of transition and intermediate states of reactions provides insights into their mechanisms and is often achieved through analysis of linear free energy relationships. Such an approach has been used extensively in protein folding studies but less so for analyzing allosteric transitions. Here, we point out analogies in ways to characterize pathways and intermediates in folding and allosteric transitions. Achieving an understanding of the mechanisms by which proteins undergo allosteric switching is important in many cases for obtaining insights into how they function.

Confining compartments are ubiquitous in biology, but there have been few experimental studies on the thermodynamics of protein folding in such environments. Recently, we reported that the stability of a model protein substrate in the GroEL/ES chaperonin cage is reduced dramatically by more than 5 kcal mol⁻¹ compared to that in bulk solution, but the origin of this effect remained unclear. Here, we show that this destabilization is caused, at least in part, by a diminished hydrophobic effect in the GroEL/ES cavity. This reduced hydrophobic effect is probably caused by water ordering due to the small number of hydration shells between the cavity and protein substrate surfaces. Hence, encapsulated protein substrates can undergo a process similar to cold denaturation in which unfolding is promoted by ordered water molecules. Our findings are likely to be relevant to encapsulated substrates in chaperonin systems, in general, and are consistent with the iterative annealing mechanism of action proposed for GroEL/ES.

Recent progress in structure-prediction methods that rely on deep learning suggests that the atomic structure of almost any protein may soon be predictable directly from its amino acid sequence. This much-awaited revolution was driven by substantial improvements in the reliability of methods for inferring the spatial distances between amino acid pairs from an analysis of homologous sequences. Improved reliability has been accompanied, however, by a reduced ability to detect amino acid relationships that are not due to direct spatial contacts, such as those that arise from protein dynamics or allostery. Given the central importance of dynamics and allostery to protein activity, we argue that an important future advance would extend modeling beyond predicting a single static structure. Here, we briefly review some of the developments that have led to the remarkable recent achievement in structure prediction and speculate what methods and sources of information may be leveraged in the future to develop a modeling framework that addresses protein dynamics and allostery.

About 20% of all familial amyotrophic lateral sclerosis (ALS) cases are associated with mutations in superoxide dismutase (SOD1), a homodimeric protein. The disease has an autosomal-dominant inheritance pattern. It is, therefore, important to determine whether wild-type and mutant SOD1 subunits self-associate randomly or preferentially. A measure for the extent of bias in subunit association is the coupling constant determined in a double-mutant cycle type analysis. Here, cell lysates containing co-expressed wild-type and mutant SOD1 subunits were analyzed by native mass spectrometry to determine these coupling constants. Strikingly, we find a linear positive correlation between the coupling constant and the reported average duration of the disease. Our results indicate that inter-subunit communication and a preference for heterodimerization greatly increase the disease severity.

Bleomycin is an anticancer antibiotic effective against a range of human malignancies. Yet its usefulness is limited by serious side effects. In this study, we converted bleomycin into a prodrug by covalently linking 2-sulfo, 9 fluorenylmethoxycarbonyl (FMS) to the primary amino side chain of bleomycin. FMS-bleomycin lost its efficacy to bind transition metal ions and therefore was converted into an inactive derivative. Upon incubation in vitro under physiological conditions, the FMS-moiety undergoes spontaneous hydrolysis, generating native bleomycin possessing full anti-bacterial potency. FMS hydrolysis and reactivation takes place with a t(1/2) value of 17 +/- 1 h. In silico simulation predicts a narrow therapeutic window in human patients of seven hours, starting 40 min after administration. In mice, close agreement was obtained between the experimental and the simulated pharmacokinetic profiles for FMS-bleomycin. FMS-bleomycin is thus shown to be a classical prodrug: it is inactive at the time of administration and the non-modified (active) bleomycin is released with a desirable pharmacokinetic profile following administration, suggesting it may have therapeutic value in the clinic.

A powerful method to determine the energetic coupling between amino acids is double mutant cycle analysis. In this method, two residues are mutated separately and in combination and the energetic effects of the mutations are determined. A deviation of the effect of the double mutation from the sum of effects of the single mutations indicates that the two residues are interacting directly or indirectly. Here, we show that double mutant cycle analysis by native mass spectrometry can be carried out for interactions in crude Escherichia coli cell extracts, thereby obviating the need for protein purification and generating binding isotherms. Our results indicate that intermolecular hydrogen bond strengths are not affected by the more crowded conditions in cell lysates.

The GroE chaperonin system in Escherichia coli comprises GroEL and GroES and facilitates ATP-dependent protein folding in vivo and in vitro Proteins with very similar sequences and structures can differ in their dependence on GroEL for efficient folding. One potential but unverified source for GroEL dependence is frustration, wherein not all interactions in the native state are optimized energetically, thereby potentiating slow folding and misfolding. Here, we chose enhanced green fluorescent protein as a model system and subjected it to random mutagenesis, followed by screening for variants whose in vivo folding displays increased or decreased GroEL dependence. We confirmed the altered GroEL dependence of these variants with in vitro folding assays. Strikingly, mutations at positions predicted to be highly frustrated were found to correlate with decreased GroEL dependence. Conversely, mutations at positions with low frustration were found to correlate with increased GroEL dependence. Further support for this finding was obtained by showing that folding of an enhanced green fluorescent protein variant designed computationally to have reduced frustration is indeed less GroEL-dependent. Our results indicate that changes in local frustration also affect partitioning in vivo between spontaneous and chaperonin-mediated folding. Hence, the design of minimally frustrated sequences can reduce chaperonin dependence and improve protein expression levels.

The chaperonin-containing t-complex polypeptide 1 (CCT, also known as TRiC) assists protein folding in an ATP-dependent manner. CCT/TRiC was mixed rapidly with different concentrations of ATP, and the amount of phosphate formed upon ATP hydrolysis was measured as a function of time using the coumarin-labeled phosphate-binding protein method. Two burst phases were observed, followed by a lag phase and then a linear steady-state phase of ATP hydrolysis. The phases were assigned by (i) determining their dependence on ATP and K+ concentrations and (ii) by measuring their sensitivity to the mutation Gly345 -> 4Asp in subunit CCT4, which decreases cooperativity in ATP binding. The values of the observed rate constants corresponding to the burst phases are found to decrease with increasing ATP and K+ concentrations, thereby indicating that the apo state of CCT/TRiC is in equilibrium between several conformations and that "conformational selection" by ATP takes place before hydrolysis. The amplitude of the lag phase, which follows, decreases with increasing ATP concentrations, thus indicating that it reflects a transition between states with low affinity for ATP and a state with high affinity for ATP that is predominant under steady-state conditions. A kinetic model based on the data is suggested, in which CCT/TRiC is in equilibrium between a relatively large number of states that are distinguished kinetically, in agreement with its proposed sequential allosteric mechanism. (C) 2016 Elsevier Ltd. All rights reserved.

The ability to query enzyme molecules individually is transforming our view of catalytic mechanisms. Quiescin sulfhydryl oxidase (QSOX) is a multidomain catalyst of disulfide-bond formation that relays electrons from substrate cysteines through two redox-active sites to molecular oxygen. The chemical steps in electron transfer have been delineated, but the conformational changes accompanying these steps are poorly characterized. Here we use single-molecule Förster resonance energy transfer (smFRET) to probe QSOX conformation in resting and cycling enzyme populations. We report the discovery of unanticipated roles for conformational changes in QSOX beyond mediating electron transfer between redox-active sites. In particular, a state of the enzyme not previously postulated or experimentally detected is shown to gate, via a conformational transition, the entrance into a sub-cycle within an expanded QSOX kinetic scheme. By tightly constraining mechanistic models, smFRET data can reveal the coupling between conformational and chemical transitions in complex enzymatic cycles.

ATP-dependent binding of the chaperonin GroEL to its cofactor GroES forms a cavity in which encapsulated substrate proteins can fold in isolation from bulk solution. It has been suggested that folding in the cavity may differ from that in bulk solution owing to steric confinement, interactions with the cavity walls, and differences between the properties of cavity-confined and bulk water. However, experimental data regarding the cavity-confined water are lacking. Here, we report measurements of water density and diffusion dynamics in the vicinity of a spin label attached to a cysteine in the Tyr71 → Cys GroES mutant obtained using two magnetic resonance techniques: electron-spin echo envelope modulation and Overhauser dynamic nuclear polarization. Residue 71 in GroES is fully exposed to bulk water in free GroES and to confined water within the cavity of the GroEL-GroES complex. Our data show that water density and translational dynamics in the vicinity of the label do not change upon complex formation, thus indicating that bulk water-exposed and cavity-confined GroES surface water share similar properties. Interestingly, the diffusion dynamics of water near the GroES surface are found to be unusually fast relative to other protein surfaces studied. The implications of these findings for chaperonin-assisted folding mechanisms are discussed.

The eukaryotic chaperonin containing t-complex polypeptide 1 (CCT/TRiC) is an ATP-fueled machine that assists protein folding. It consists of two back-to-back stacked rings formed by eight different subunits that are arranged in a fixed permutation. The different subunits of CCT are believed to possess unique substrate binding specificities that are still mostly unknown. Here, we used high-throughput microscopy analysis of yeast cells to determine changes in protein levels and localization as a result of a Glu to Asp mutation in the ATP binding site of subunits 3 (CCT3) or 6 (CCT6). The mutation in subunit CCT3 was found to induce cytoplasmic foci termed P-bodies where mRNAs, which are not translated, accumulate and can be degraded. Analysis of the changes in protein levels and structural modeling indicate that P-body formation in cells with the mutation in CCT3 is linked to the specific interaction of this subunit with Gln/Asn-rich segments that are enriched in many P-body proteins. An in vitro gel-shift analysis was used to show that the mutation in subunit CCT3 interferes with the ability of CCT to bind a Gln/Asn-rich protein aggregate. More generally, the strategy used in this work can be used to unravel the substrate specificities of other chaperone systems.

Chaperonins such as GroEL from Escherichia coli are molecular machines that facilitate protein folding by undergoing energy (ATP)-dependent movements that are coordinated in time and space owing to complex allosteric regulation. Here, we describe some of the various functional (allosteric) states of GroEL, the pathways by which they inter-convert and the coupling between allosteric transitions and protein folding reactions.

There is often an interest in knowing, for a given ligand concentration, how many protein molecules have one, two, three, etc. ligands bound in a specific manner. This is a question that cannot be addressed using conventional ensemble techniques. Here, a mathematical method is presented for separating specific from nonspecific binding in nonensemble studies. The method provides a way to determine the distribution of specific binding stoichiometries at any ligand concentration when using nonensemble (e.g., single-molecule) methods. The applicability of the method is demonstrated for ADP binding to creatine kinase using mass spectroscopy data. A major advantage of our method, which can be applied to any protein-ligand system, is that no previous information regarding the mechanism of ligand interaction is required.

Two different strategies for stabilizing proteins are (i) positive design in which the native state is stabilized and (ii) negative design in which competing non-native conformations are destabilized. Here, the circumstances under which one strategy might be favored over the other are explored in the case of lattice models of proteins and then generalized and discussed with regard to real proteins. The balance between positive and negative design of proteins is found to be determined by their average "contact- frequency", a property that corresponds to the fraction of states in the conformational ensemble of the sequence in which a pair of residues is in contact. Lattice model proteins with a high average contact-frequency are found to use negative design more than model proteins with a low average contact-frequency. A mathematical derivation of this result indicates that it is general and likely to hold also for real proteins. Comparison of the results of correlated mutation analysis for real proteins with typical contact-frequencies to those of proteins likely to have high contactfrequencies (such as disordered proteins and proteins that are dependent on chaperonins for their folding) indicates that the latter tend to have stronger interactions between residues that are not in contact in their native conformation. Hence, our work indicates that negative design is employed when insufficient stabilization is achieved via positive design owing to high contact-frequencies.

We describe the design of an optical switch in the chaperonin GroEL that is opened and closed by its ATP- and cochaperonin GroES-driven conformational changes. The switch, based on a fluorophore and a quencher, is engineered into the single-ring variant of the chaperone, and shows dramatic modulation of its fluorescent intensity in response to the transition of the protein between its allosteric states. It, therefore, forms a sensitive probe for the dynamics of the allosteric transitions of this machine, both in the bulk and in single molecules.

Motivation: Theoretical considerations have indicated that the amount of chaperonin GroEL in Escherichia coli cells is sufficient to fold only ∼5° of newly synthesized proteins under normal physiological conditions, thereby suggesting that only a subset of E.coli proteins fold in vivo in a GroEL-dependent manner. Recently, members of this subset were identified in two independent studies that resulted in two partially overlapping lists of GroEL-interacting proteins. The objective of the work described here was to identify sequence-based features of GroEL-interacting proteins that distinguish them from other E.coli proteins and that may account for their dependence on the chaperonin system. Results: Our analysis shows that GroEL-interacting proteins have, on average, low folding propensities and high translation efficiencies. These two properties in combination can increase the risk of aggregation of these proteins and, thus, cause their folding to be chaperonin-dependent. Strikingly, we find that these properties are absent in proteins homologous to the E.coli GroEL-interacting proteins in Ureaplasma urealyticum, an organism that lacks a chaperonin system, thereby confirming our conclusions.

The ATPase activity of many types of molecular chaperones is stimulated by polypeptide substrate binding via molecular mechanisms that are, for the most part, unknown. Here, we report that such stimulation of the ATPase activity of GroEL is abolished when its conserved apical domain residue Glu257 is replaced by alanine. This mutation is also found to convert the ATPase profile of GroEL, a group I chaperonin, into one that is characteristic of group II chaperonins. Steady-state and transient kinetic analysis indicate that both effects are due, at least in part, to a reduction of the affinity of GroEL for ADP. This finding indicates that nonfolded proteins stimulate ATP hydrolysis by accelerating the off-rate of the ADP formed, thereby allowing more rapid cycles of ATP binding and hydrolysis.

Chaperonins are molecular machines that facilitate protein folding by undergoing energy (ATP)-dependent movements that are coordinated in time and space by complex allosteric regulation. Recently, progress has been made in describing the various functional (allosteric) states of these machines, the pathways by which they interconvert, and the coupling between allosteric transitions and protein folding reactions. However, various mechanistic issues remain to be resolved.

The reaction cycle of the double-ring chaperonin GroEL is driven by ATP binding that takes place with positive cooperativity within each seven-membered ring and negative cooperativity between rings. The positive cooperativity within rings is due to ATP binding-induced conformational changes that are fully concerted. Herein, it is shown that the mutation Asp-155 → Ala leads to an ATP-induced break in intra-ring and inter-ring symmetry. Electron microscopy analysis of single-ring GroEL particles containing the Asp-155 → Ala mutation shows that the break in intra-ring symmetry is due to stabilization of allosteric intermediates such as one in which three subunits have switched their conformation while the other four have not. Our results show that eliminating an intra-subunit interaction between Asp-155 and Arg-395 results in conversion of the allosteric switch of GroEL from concerted to sequential, thus demonstrating that its allosteric behavior arises from coupled tertiary conformational changes.

What are the mechanisms of ligand-induced allosteric transitions in proteins? A powerful method to characterize pathways and transition states of reactions is φ value analysis. A φ value is the ratio between the changes on a perturbation (e.g., mutation) in the activation and equilibrium free energies of a reaction. Here, φ value analysis is used to characterize the ATP-induced allosteric transitions of GroEL by using changes in ATP concentration as perturbations. GroEL consists of two stacked back-to-back heptameric rings that bind ATP with positive cooperativity within rings and negative cooperativity between rings. Evidence is presented for the existence of parallel pathways for the allosteric transition of each ring. In both allosteric transitions, there is an abrupt ATP-dependent switch from a pathway with ATP-binding sites in the transition state that are very similar to those of the initial T state (φ = 0) to a pathway with a φ value of ≡0.3. The φ value procedure outlined here should be useful in mapping the energy landscape of allosteric transitions of other proteins.

Initial rates of ATP hydrolysis by the chaperonin containing TCP-1 (CCT) from bovine testis were measured as a function of ATP concentration. Two allosteric transitions are observed: one at relatively low concentrations of ATP (

Escherichia coli cells that produce only plasmid-encoded wild-type or mutant GroEL were generated by bacteriophage P1 transduction. Effects of mutations that affect the allosteric properties of GroEL were characterized in vivo. Cells containing only GroEL(R197A), which has reduced intra-ring positive cooperativity and inter-ring negative cooperativity in ATP binding, grow poorly upon a temperature shift from 25 to 42 °C. This strain supports the growth of phages T4 and T5 but not phage λ and produces light at 28°C when transformed with a second plasmid containing the lux operon. In contrast, cells containing only GroEL(R13G, A126V) which lacks negative cooperativity between rings but has intact intra-ring positive cooperativity grow normally and support phage growth but do not produce light at 28 °C. In vitro refolding of luciferase in the presence of this mutant is found to be less efficient compared with wild-type GroEL or other mutants tested. Our results show that allostery in GroEL is important in vivo in a manner that depends on the physiological conditions and is protein substrate specific.

The Fv fragment of the 0.5β monoclonal antibody has recently been constructed, expressed, and purified. It binds with nanomolar affinity to the immunogenic RP185 peptide that is derived from the principal neutralizing determinant of HIV-1 in the third hypervariable region of gp120. Here, we analyzed the temperature-dependence of binding of the 0.5β Fv fragment to the RP135 peptide and a series of mutants thereof. Our results show that there is almost complete enthalpyentropy compensation in the effects of mutations in the peptide on binding to the Fv, indicating that the mutations do not change the binding mechanism. There is good correlation, for residues within the antigenic epitope, between mutational effects on ΔC(p) and calculated values of ΔDC(p) based on the extent of burial of polar and non- polar surfaces areas of amino acids. The value of ΔC(p) for the binding of the 0.5β Fv fragment to the wild-type RP135 peptide is found to be -5.0 (±0.9) kcal K^-1 mol^-1 in the presence of 0.1% Tween-20 but only -0.1 (±0.9) kcal K^-1 mol^-1 in its absence. This result has important implications for the successful application of the structural parameterization approach to predicting changes in heat capacity that accompany binding reactions carried out in the presence of detergent or protein-stabilizing agents.

The construction, expression, and purification of an active Fv fragment of the 0.5β monoclonal human immunodeficiency virus type 1 (HIV-1) neutralizing antibody is reported. The interaction between the Fv fragment and the RP135 peptide derived from the V3 loop of gp120 from HIV-1(IIIB) was studied by varying the salt concentration and by mutating arginine residues in the peptide. The mutations R4A, R8A and R11A (which correspond to residues 311,315, and 318 in gp120 of HIV-1(IIIB)) reduce the binding free energy by 0.22 (± 0.20), 4.32 (± 0.16), and 1.58 (± 0.17) kcal mol^-1, respectively. The salt-dependent components of their contributions to binding are 0.02 (± 0.22), -0.55 (± 0.18), and -0.97 (± 0.19) kcal mol^-1, respectively. The magnitudes of the mutational effects and the extent of shielding by 1 M NaCl suggest that Arg-8 is involved in a buried salt bridge in the peptide-Fv fragment complex, whereas Arg-11 is involved in a more solvent-exposed electrostatic interaction.

Initial rates of ATP hydrolysis by wild-type GroEL were measured as a function of ATP concentration from 0 to 0.8 mM. Two allosteric transitions are observed: one at relatively low ATP concentrations (⩽ 100μM) and the second at higher concentrations of ATP with respective midpoints of about 16 and 160μM. Two allosteric transitions were previously observed also in the case of the Arg- 196 → Ala GroEL mutant [Yifrach, O., & Horovitz, A. (1994) J. Mol. Biol. 243, 397-401], On the basis of these observations a mathematical model for nested cooperativity in ATP hydrolysis by GroEL is developed in which there are two levels of allostery: one within each ring and the second between rings. In the first level, each heptameric ring is in equilibrium between the T and R states, in accordance with the Monod-Wyman-Changeux (MWC) model of cooperativity [Monod et al. (1965) J. Mol. Biol. 12, 88-118], A second level of allostery is between the rings of the GroEL particle which undergoes sequential Koshland-Nemethy-Filmer (KNF)-type transitions from the TT state via the TR state to the RR state [Koshland et al. (1966) Biochemistry 5, 365-385]. Using our model, we estimate the values of the Hill coefficient for the negative cooperativity between rings in wild-type GroEL and the Arg-196 → Ala mutant to be 0.003 (±0.001) and 0.07 (±0.02), respectively. The inter-ring coupling free energies in wild-type GroEL and the Arg-196 - Ala mutant are -7.5 (±0.4) and -3.9 (±0.3) kcal mol^-1, respectively.

The conserved residue Lys-34 in GroES was replaced by alanine and glutamic acid using site-directed mutagenesis. This residue is near the carboxy terminus of the mobile loop in GroES (residues 17-32) which becomes immobilized upon formation of the GroEL/GroES complex [Landry et al. (1993) Nature 364, 255-258]. Both charge neutralization (Lys-34-->Ala) and charge reversal (Lys-34--->Glu) at this position have little effect on the binding constant of GroES to GroEL, but they increase the enhancement by GroES of cooperativity in ATP hydrolysis by GroEL. This is reflected by a change in the Hill coefficient (at 10 mM K+) from 4.10 (+/-0.22) in the presence of wild-type GroES to 5.17 (+/-0.24) and 4.46 (+/-0.14) in the presence of the GroES mutants Lys-34-->Ala and Lys-34-->Glu, respectively. The results are interpreted using the Monod-Wyman-Changeux (MWC) model for cooperativity [Monod et al. (1965) J. Mol. Biol. 12, 88-118]. They suggest that Lys-34 in GroES modulates the allosteric transition in GroEL by stabilizing a relaxed (R)-like state.

The Escherichia coli heat-shock protein GroEL is a member of the highly conserved family of tetradecameric chaperonins 60, which assist in the folding and assembly of other proteins. Using site-directed mutagenesis, it is shown that replacement of the absolutely conserved amino acid residue Lys-3 by arginine or isoleucine destabilizes the GroEL particle and that the replacement Lys-3 → Glu completely blocks its formation. The rank order of effects of these mutations on the stability of the GroEL particle correlates with the associated changes in net charge at that position. Our results show that the N terminus of GroEL is a crucial structural element for its assembly.