Sensing cooperativity in ATP hydrolysis for single multisubunit enzymes in solution.

In order to operate in a coordinated fashion, multisubunit enzymes use cooperative interactions intrinsic to their enzymatic cycle, but this process remains poorly understood. Accordingly, ATP number distributions in various hydrolyzed states have been obtained for single copies of the mammalian double-ring multisubunit chaperonin TRiC/CCT in free solution using the emission from chaperonin-bound fluorescent nucleotides and closed-loop feedback trapping provided by an Anti-Brownian ELectrokinetic trap. Observations of the 16-subunit complexes as ADP molecules are dissociating shows a peak in the bound ADP number distribution at 8 ADP, whose height falls over time with little shift in the position of the peak, indicating a highly cooperative ADP release process which would be difficult to observe by ensemble-averaged methods. When AlFx is added to produce ATP hydrolysis transition state mimics (ADP·AlFx) locked to the complex, the peak at 8 nucleotides dominates for all but the lowest incubation concentrations. Although ensemble averages of the single-molecule data show agreement with standard cooperativity models, surprisingly, the observed number distributions depart from standard models, illustrating the value of these single-molecule observations in constraining the mechanism of cooperativity. While a complete alternative microscopic model cannot be defined at present, the addition of subunit-occupancy-dependent cooperativity in hydrolysis yields distributions consistent with the data.

Action of the chaperonin GroEL/ES on a non-native substrate observed with single-molecule FRET.

The double ring-shaped chaperonin GroEL binds a wide range of non-native polypeptides within its central cavity and, together with its cofactor GroES, assists their folding in an ATP-dependent manner. The conformational cycle of GroEL/ES has been studied extensively but little is known about how the environment in the central cavity affects substrate conformation. Here, we use the von Hippel-Lindau tumor suppressor protein VHL as a model substrate for studying the action of the GroEL/ES system on a bound polypeptide. Fluorescent labeling of pairs of sites on VHL for fluorescence (Förster) resonant energy transfer (FRET) allows VHL to be used to explore how GroEL binding and GroEL/ES/nucleotide binding affect the substrate conformation. On average, upon binding to GroEL, all pairs of labeling sites experience compaction relative to the unfolded protein while single-molecule FRET distributions show significant heterogeneity. Upon addition of GroES and ATP to close the GroEL cavity, on average further FRET increases occur between the two hydrophobic regions of VHL, accompanied by FRET decreases between the N- and C-termini. This suggests that ATP- and GroES-induced confinement within the GroEL cavity remodels bound polypeptides by causing expansion (or racking) of some regions and compaction of others, most notably, the hydrophobic core. However, single-molecule observations of the specific FRET changes for individual proteins at the moment of ATP/GroES addition reveal that a large fraction of the population shows the opposite behavior; that is, FRET decreases between the hydrophobic regions and FRET increases for the N- and C-termini. Our time-resolved single-molecule analysis reveals the underlying heterogeneity of the action of GroES/EL on a bound polypeptide substrate, which might arise from the random nature of the specific binding to the various identical subunits of GroEL, and might help explain why multiple rounds of binding and hydrolysis are required for some chaperonin substrates.

4.0-A resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement.

The essential double-ring eukaryotic chaperonin TRiC/CCT (TCP1-ring complex or chaperonin containing TCP1) assists the folding of approximately 5-10% of the cellular proteome. Many TRiC substrates cannot be folded by other chaperonins from prokaryotes or archaea. These unique folding properties are likely linked to TRiC's unique heterooligomeric subunit organization, whereby each ring consists of eight different paralogous subunits in an arrangement that remains uncertain. Using single particle cryo-EM without imposing symmetry, we determined the mammalian TRiC structure at 4.7-A resolution. This revealed the existence of a 2-fold axis between its two rings resulting in two homotypic subunit interactions across the rings. A subsequent 2-fold symmetrized map yielded a 4.0-A resolution structure that evinces the densities of a large fraction of side chains, loops, and insertions. These features permitted unambiguous identification of all eight individual subunits, despite their sequence similarity. Independent biochemical near-neighbor analysis supports our cryo-EM derived TRiC subunit arrangement. We obtained a Calpha backbone model for each subunit from an initial homology model refined against the cryo-EM density. A subsequently optimized atomic model for a subunit showed approximately 95% of the main chain dihedral angles in the allowable regions of the Ramachandran plot. The determination of the TRiC subunit arrangement opens the way to understand its unique function and mechanism. In particular, an unevenly distributed positively charged wall lining the closed folding chamber of TRiC differs strikingly from that of prokaryotic and archaeal chaperonins. These interior surface chemical properties likely play an important role in TRiC's cellular substrate specificity.

The folding trajectory of RNase H is dominated by its topology and not local stability: a protein engineering study of variants that fold via two-state and three-state mechanisms.

Proteins can sample a variety of partially folded conformations during the transition between the unfolded and native states. Some proteins never significantly populate these high-energy states and fold by an apparently two-state process. However, many proteins populate detectable, partially folded forms during the folding process. The role of such intermediates is a matter of considerable debate. A single amino acid change can convert Escherichia coli ribonuclease H from a three-state folder that populates a kinetic intermediate to one that folds in an apparent two-state fashion. We have compared the folding trajectories of the three-state RNase H and the two-state RNase H, proteins with the same native-state topology but altered regional stability, using a protein engineering approach. Our data suggest that both versions of RNase H fold through a similar trajectory with similar high-energy conformations. Mutations in the core and the periphery of the protein affect similar aspects of folding for both variants, suggesting a common trajectory with folding of the core region followed by the folding of the periphery. Our results suggest that formation of specific partially folded conformations may be a general feature of protein folding that can promote, rather than hinder, efficient folding.

Identification of the TRiC/CCT substrate binding sites uncovers the function of subunit diversity in eukaryotic chaperonins.

The ring-shaped hetero-oligomeric chaperonin TRiC/CCT uses ATP to fold a diverse subset of eukaryotic proteins. To define the basis of TRiC/CCT substrate recognition, we mapped the chaperonin interactions with the VHL tumor suppressor. VHL has two well-defined TRiC binding determinants. Each determinant contacts a specific subset of chaperonin subunits, indicating that TRiC paralogs exhibit distinct but overlapping specificities. The substrate binding site in these subunits localizes to a helical region in the apical domains that is structurally equivalent to that of bacterial chaperonins. Transferring the distal portion of helix 11 between TRiC subunits suffices to transfer specificity for a given substrate motif. We conclude that the architecture of the substrate binding domain is evolutionarily conserved among eukaryotic and bacterial chaperonins. The unique combination of specificity and plasticity in TRiC substrate binding may diversify the range of motifs recognized by this chaperonin and contribute to its unique ability to fold eukaryotic proteins.

Modeling of possible subunit arrangements in the eukaryotic chaperonin TRiC.

The eukaryotic cytosolic chaperonin TRiC (TCP-1 Ring Complex), also known as CCT (Cytosolic Chaperonin containing TCP-1), is a hetero-oligomeric complex consisting of two back-to-back rings of eight different subunits each. The general architecture of the complex has been determined, but the arrangement of the subunits within the complex remains an open question. By assuming that the subunits have a defined arrangement within each ring, we constructed a simple model of TRiC that analyzes the possible arrangements of individual subunits in the complex. By applying the model to existing data, we find that there are only four subunit arrangements consistent with previous observations. Our analysis provides a framework for the interpretation and design of experiments to elucidate the quaternary structure of TRiC/CCT. This in turn will aid in the understanding of substrate binding and allosteric properties of this chaperonin.

Protein folding: defining a "standard" set of experimental conditions and a preliminary kinetic data set of two-state proteins.

Recent years have seen the publication of both empirical and theoretical relationships predicting the rates with which proteins fold. Our ability to test and refine these relationships has been limited, however, by a variety of difficulties associated with the comparison of folding and unfolding rates, thermodynamics, and structure across diverse sets of proteins. These difficulties include the wide, potentially confounding range of experimental conditions and methods employed to date and the difficulty of obtaining correct and complete sequence and structural details for the characterized constructs. The lack of a single approach to data analysis and error estimation, or even of a common set of units and reporting standards, further hinders comparative studies of folding. In an effort to overcome these problems, we define here a "consensus" set of experimental conditions (25 degrees C at pH 7.0, 50 mM buffer), data analysis methods, and data reporting standards that we hope will provide a benchmark for experimental studies. We take the first step in this initiative by describing the folding kinetics of 30 apparently two-state proteins or protein domains under the consensus conditions. The goal of our efforts is to set uniform standards for the experimental community and to initiate an accumulating, self-consistent data set that will aid ongoing efforts to understand the folding process.

Destabilization of the Escherichia coli RNase H kinetic intermediate: switching between a two-state and three-state folding mechanism.

Escherichia coli RNase H folds through a partially folded kinetic intermediate that mirrors a rarely populated, partially unfolded form detectable by native-state hydrogen exchange under equilibrium conditions. Residue 53 is at the interface of two helices known to be structured in this intermediate. Kinetic refolding studies on mutant proteins varying in size and hydrophobicity at residue 53 support a contribution of hydrophobicity to the stabilities of the kinetic intermediate and the transition state. Packing interactions also play a significant role in the stability of these two states, though they play a much larger role in the native-state stability. One dramatic mutation, I53D, results in the conversion from a three-state to a two-state folding mechanism, which is explained most easily through a simple destabilization of the kinetic intermediate such that it is no longer stable with respect to the unfolded state. These results demonstrate that interactions that stabilize an intermediate can accelerate folding if these same interactions are present in the transition state. Our results are consistent with a hierarchical model of folding, where the intermediate consists of native-like interactions, is on-pathway, and is productive for folding.

Experimental evaluation of topological parameters determining protein-folding rates.

Recent work suggests that structural topology plays a key role in determining protein-folding rates and pathways. The refolding rates of small proteins that fold without intermediates are found to correlate with simple structural parameters such as relative contact order, long-range order, or the fraction of short-range contacts. To test and evaluate the role of structural topology experimentally, a set of circular permutants of the ribosomal protein S6 from Thermus thermophilus was analyzed. Despite a wide range of relative contact order, the permuted proteins all fold with similar rates. These results suggest that alternative topological parameters may better describe the role of topology in protein-folding rates.