A diminished hydrophobic effect inside the GroEL/ES cavity contributes to protein substrate destabilization.

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.

Cooperativity in ATP Hydrolysis by MopR Is Modulated by Its Signal Reception Domain and by Its Protein and Phenol Concentrations.

The NtrC family of AAA+ proteins are bacterial transcriptional regulators that control σ54-dependent RNA polymerase transcription under certain stressful conditions. MopR, which is a member of this family, is responsive to phenol and stimulates its degradation. Biochemical studies to understand the role of ATP and phenol in oligomerization and allosteric regulation, which are described here, show that MopR undergoes concentration-dependent oligomerization in which dimers assemble into functional hexamers. The oligomerization occurs in a nucleation-dependent manner with a tetrameric intermediate. Additionally, phenol binding is shown to be responsible for shifting MopR's equilibrium from a repressed state (high affinity toward ATP) to a functionally active, derepressed state with low-affinity for ATP. Based on these findings, we propose a model for allosteric regulation of MopR. IMPORTANCE The NtrC family of bacterial transcriptional regulators are enzymes with a modular architecture that harbor a signal sensing domain followed by a AAA+ domain. MopR, a NtrC family member, responds to phenol and activates phenol adaptation pathways that are transcribed by σ54-dependent RNA polymerases. Our results show that for efficient ATP hydrolysis, MopR assembles as functional hexamers and that this activity of MopR is regulated by its effector (phenol), ATP, and protein concentration. Our findings, and the kinetic methods we employ, should be useful in dissecting the allosteric mechanisms of other AAA+ proteins, in general, and NtrC family members in particular.

Intracellular Protein-Drug Interactions Probed by Direct Mass Spectrometry of Cell Lysates.

Understanding protein-ligand interactions in a cellular context is an important goal in molecular biology and biochemistry, and particularly for drug development. Investigators must demonstrate that drugs penetrate cells and specifically bind their targets. Towards that end, we present a native mass spectrometry (MS)-based method for analyzing drug uptake and target engagement in eukaryotic cells. This method is based on our previously introduced direct-MS method for rapid analysis of proteins directly from crude samples. Here, direct-MS enables label-free studies of protein-drug binding in human cells and is used to determine binding affinities of lead compounds in crude samples. We anticipate that this method will enable the application of native MS to a range of problems where cellular context is important, including protein-protein interactions, drug uptake and binding, and characterization of therapeutic proteins.

Reconciling the controversy regarding the functional importance of bullet- and football-shaped GroE complexes.

The chaperonin GroEL and its co-chaperonin GroES form both GroEL-GroES bullet-shaped and GroEL-GroES2 football-shaped complexes. The residence time of protein substrates in the cavities of these complexes is about 10 and 1 s, respectively. There has been much controversy regarding which of these complexes is the main functional form. Here, we show using computational analysis that GroEL protein substrates have a bimodal distribution of folding times, which matches these residence times, thereby suggesting that both bullet-shaped and football-shaped complexes are functional. More generally, co-existing complexes with different stoichiometries are not mutually exclusive with respect to having a functional role and can complement each other.

Sequential allosteric mechanism of ATP hydrolysis by the CCT/TRiC chaperone is revealed through Arrhenius analysis.

Knowing the mechanism of allosteric switching is important for understanding how molecular machines work. The CCT/TRiC chaperonin nanomachine undergoes ATP-driven conformational changes that are crucial for its folding function. Here, we demonstrate that insight into its allosteric mechanism of ATP hydrolysis can be achieved by Arrhenius analysis. Our results show that ATP hydrolysis triggers sequential ?conformational waves." They also suggest that these waves start from subunits CCT6 and CCT8 (or CCT3 and CCT6) and proceed clockwise and counterclockwise, respectively.

GroEL Allostery Illuminated by a Relationship between the Hill Coefficient and the MWC Model.

A fundamental problem that has hindered the use of the classic Monod-Wyman-Changuex (MWC) allosteric model since its introduction is that it has been difficult to determine the values of its parameters in a reliable manner because they are correlated with each other and sensitive to the data-fitting method. Consequently, experimental data are often fitted to the Hill equation, which provides a measure of cooperativity but no insights into its origin. In this work, we derived a general relationship between the value of the Hill coefficient and the parameters of the MWC model. It is shown that this relationship can be used to select the best estimate of the true combination of the MWC parameter values from all the possible ones found to fit the data. Here, this approach was applied to fits to the MWC model of curves of the fraction of GroEL molecules in the high-affinity (R) state for ATP as a function of ATP concentration. Such curves were collected at different temperatures, thereby providing insight into the hydrophobic effect associated with the ATP-promoted allosteric switch of GroEL. More generally, the relationship derived here should facilitate future thermodynamic analysis of other MWC-type allosteric systems.

Contact Order Is a Determinant for the Dependence of GFP Folding on the Chaperonin GroEL.

The GroE chaperonin system facilitates protein folding in an ATP-dependent manner. It has remained unclear why some proteins are obligate clients of the GroE system, whereas other closely related proteins are able to fold efficiently in its absence. Factors that cause folding to be slower affect kinetic partitioning between spontaneous folding and chaperone binding in favor of the latter. One such potential factor is contact order (CO), which is the average separation in sequence between residues that are in contact in the native structure. Here, we generated variants of enhanced green fluorescent protein with different COs using circular permutations. We found that GroE dependence in vitro and in vivo increases with increasing CO. Thus, our results show that CO is relevant not only for folding in vitro of relatively simple model systems but also for chaperonin dependence and folding in vivo.

Converting bleomycin into a prodrug that undergoes spontaneous reactivation under physiological conditions.

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 t1/2 value of 17 ±â€¯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.

Local energetic frustration affects the dependence of green fluorescent protein folding on the chaperonin GroEL.

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.

Estimating Interprotein Pairwise Interaction Energies in Cell Lysates from a Single Native Mass Spectrum.

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.

Allosteric Mechanisms in Chaperonin Machines.

Chaperonins are nanomachines that facilitate protein folding by undergoing energy (ATP)-dependent movements that are coordinated in time and space owing to complex allosteric regulation. They consist of two back-to-back stacked oligomeric rings with a cavity at each end where protein substrate folding can take place. Here, we focus on the GroEL/GroES chaperonin system from Escherichia coli and, to a lesser extent, on the more poorly characterized eukaryotic chaperonin CCT/TRiC. We describe their various functional (allosteric) states and how they are affected by substrates and allosteric effectors that include ATP, ADP, nonfolded protein substrates, potassium ions, and GroES (in the case of GroEL). We also discuss the pathways of intra- and inter-ring allosteric communication by which they interconvert and the coupling between allosteric transitions and protein folding reactions.

Comparative study of the effectiveness and limitations of current methods for detecting sequence coevolution.

MOTIVATION: With rapid accumulation of sequence data on several species, extracting rational and systematic information from multiple sequence alignments (MSAs) is becoming increasingly important. Currently, there is a plethora of computational methods for investigating coupled evolutionary changes in pairs of positions along the amino acid sequence, and making inferences on structure and function. Yet, the significance of coevolution signals remains to be established. Also, a large number of false positives (FPs) arise from insufficient MSA size, phylogenetic background and indirect couplings. RESULTS: Here, a set of 16 pairs of non-interacting proteins is thoroughly examined to assess the effectiveness and limitations of different methods. The analysis shows that recent computationally expensive methods designed to remove biases from indirect couplings outperform others in detecting tertiary structural contacts as well as eliminating intermolecular FPs; whereas traditional methods such as mutual information benefit from refinements such as shuffling, while being highly efficient. Computations repeated with 2,330 pairs of protein families from the Negatome database corroborated these results. Finally, using a training dataset of 162 families of proteins, we propose a combined method that outperforms existing individual methods. Overall, the study provides simple guidelines towards the choice of suitable methods and strategies based on available MSA size and computing resources. AVAILABILITY AND IMPLEMENTATION: Software is freely available through the Evol component of ProDy API.

Allosteric mechanisms can be distinguished using structural mass spectrometry.

The activity of many proteins, including metabolic enzymes, molecular machines, and ion channels, is often regulated by conformational changes that are induced or stabilized by ligand binding. In cases of multimeric proteins, such allosteric regulation has often been described by the concerted Monod-Wyman-Changeux and sequential Koshland-Némethy-Filmer classic models of cooperativity. Despite the important functional implications of the mechanism of cooperativity, it has been impossible in many cases to distinguish between these various allosteric models using ensemble measurements of ligand binding in bulk protein solutions. Here, we demonstrate that structural MS offers a way to break this impasse by providing the full distribution of ligand-bound states of a protein complex. Given this distribution, it is possible to determine all the binding constants of a ligand to a highly multimeric cooperative system, and thereby infer its allosteric mechanism. Our approach to the dissection of allosteric mechanisms relies on advances in MS--which provide the required resolution of ligand-bound states--and in data analysis. We validated our approach using the well-characterized Escherichia coli chaperone GroEL, a double-heptameric ring containing 14 ATP binding sites, which has become a paradigm for molecular machines. The values of the 14 binding constants of ATP to GroEL were determined, and the ATP-loading pathway of the chaperone was characterized. The methodology and analyses presented here are directly applicable to numerous other cooperative systems and are therefore expected to promote further research on allosteric systems.

Thermodynamic Protein Destabilization by GFP Tagging: A Case of Interdomain Allostery.

The Engrailed Homeodomain (EnHD) transcription factor of Drosophila melanogaster was fused to the enhanced green fluorescent protein (eGFP) either at its C- or N-terminus via three- or ten-residue flexible linkers. Here, we show that EnHD undergoes destabilization upon fusing it to eGFP regardless of the linker length used and whether the tethering is to its N- or C-terminus. The destabilization is reflected in melting points that are lower by up to 9°C. Thermodynamic analysis and coarse-grained molecular dynamic simulations indicate that this destabilization is due to eGFP-promoted entropic stabilization of the denatured state ensemble of EnHD. Our results provide, therefore, an example for destabilizing interdomain allostery. They are also important given the widespread use of eGFP tagging in cell biology, as they indicate that such tagging can cause unintended protein destabilization and concomitant effects.

Allosteric inhibition of individual enzyme molecules trapped in lipid vesicles.

Enzymatic inhibition by product molecules is an important and widespread phenomenon. We describe an approach to study product inhibition at the single-molecule level. Individual HRP molecules are trapped within surface-tethered lipid vesicles, and their reaction with a fluorogenic substrate is probed. While the substrate readily penetrates into the vesicles, the charged product (resorufin) gets trapped and accumulates inside the vesicles. Surprisingly, individual enzyme molecules are found to stall when a few tens of product molecules accumulate. Bulk enzymology experiments verify that the enzyme is noncompetitively inhibited by resorufin. The initial reaction velocity of individual enzyme molecules and the number of product molecules required for their complete inhibition are broadly distributed and dynamically disordered. The two seemingly unrelated parameters, however, are found to be substantially correlated with each other in each enzyme molecule and over long times. These results suggest that, as a way to counter disorder, enzymes have evolved the means to correlate fluctuations at structurally distinct functional sites.

Interactions of subunit CCT3 in the yeast chaperonin CCT/TRiC with Q/N-rich proteins revealed by high-throughput microscopy analysis.

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.

Probing water density and dynamics in the chaperonin GroEL cavity.

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.

Out-of-equilibrium conformational cycling of GroEL under saturating ATP concentrations.

The molecular chaperone GroEL exists in at least two allosteric states, T and R, that interconvert in an ATP-controlled manner. Thermodynamic analysis suggests that the T-state population becomes negligible with increasing ATP concentrations, in conflict with the requirement for conformational cycling, which is essential for the operation of molecular machines. To solve this conundrum, we performed fluorescence correlation spectroscopy on the single-ring version of GroEL, using a fluorescent switch recently built into its structure, which turns "on," i.e., increases its fluorescence dramatically, when ATP is added. A series of correlation functions was measured as a function of ATP concentration and analyzed using singular-value decomposition. The analysis assigned the signal to two states whose dynamics clearly differ. Surprisingly, even at ATP saturation, approximately 50% of the molecules still populate the T state at any instance of time, indicating constant out-of-equilibrium cycling between T and R. Only upon addition of the cochaperonin GroES does the T-state population vanish. Our results suggest a model in which the T/R ratio is controlled by the rate of ADP release after hydrolysis, which can be determined accordingly.

Distinguishing between concerted, sequential and barrierless conformational changes: Folding versus allostery.

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.

Reduced ADP off-rate by the yeast CCT2 double mutation T394P/R510H which causes Leber congenital amaurosis in humans.

The CCT/TRiC chaperonin is found in the cytosol of all eukaryotic cells and assists protein folding in an ATP-dependent manner. The heterozygous double mutation T400P and R516H in subunit CCT2 is known to cause Leber congenital amaurosis (LCA), a hereditary congenital retinopathy. This double mutation also renders the function of subunit CCT2, when it is outside of the CCT/TRiC complex, to be defective in promoting autophagy. Here, we show using steady-state and transient kinetic analysis that the corresponding double mutation in subunit CCT2 from Saccharomyces cerevisiae reduces the off-rate of ADP during ATP hydrolysis by CCT/TRiC. We also report that the ATPase activity of CCT/TRiC is stimulated by a non-folded substrate. Our results suggest that the closed state of CCT/TRiC is stabilized by the double mutation owing to the slower off-rate of ADP, thereby impeding the exit of CCT2 from the complex that is required for its function in autophagy.