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 Reprint requests to: Kevin W. Plaxco, Department of Chemistry and Biochemistry, University of California, Santa Barbara, Santa Barbara, CA 93106, USA; e-mail: kwp@chem.ucsb.edu; fax: (805) 893-4120.Abbreviations: GuHCl, guanidine hydrochloride; tris, tris hydroxymethylaminoethane; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; TCEP, tris(2-carboxyethyl)phosphine; CD, circular dichroism. Article published online ahead of print. Article and publication date are at

The essential double-ring eukaryotic chaperonin TRiC/CCT (TCP1-ring complex or chaperonin containing TCP1) assists the folding of ∼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-Å 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-Å 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 Cα 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 ∼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.asymmetric reconstruction | atomic model | subunit structure

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.

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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 chaperoninbound 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.single molecule | allostery | fluorescence | enzymology | nucleotide counting Y ears of study of cooperativity for proteins with multiple ligand binding sites have yielded important insights, ever since the role of cooperative oxygen binding to hemoglobin in high oxygendelivery throughput was recognized (1-3). Multisubunit enzymes usually hydrolyze ATP in a concerted fashion, but actually observing this process, enzyme by enzyme, can provide a deeper picture of the underlying cooperativity resulting from communication among the various subunits. A well known example has been the study of the rotary motor F1-ATPase by single-molecule techniques (4). Another class of multisubunit cellular machines is the double-ring type I and type II chaperonins which can have from 14 to 18 subunits each of which can hydrolyze ATP (5, 6). Ensemble studies of the bacterial type I GroEL/GroES system, for example, have indicated that ATP binds subunits in one 7-membered ring with positive cooperativity, while negative cooperativity operates between rings (7). We focus on the cooperative process operative in the mammalian type II chaperonin TRiC/CCT, which has two ring-shaped cavities with built-in lids composed of eight different subunits each. TRiC is essential for the folding of a number of key proteins in mammalian cells, including actin, tubulin, and many cell cycle regulators (8). Previous ensemble measurements of ATP-induced allosteric transition rates and steady-state ATPase rate in TRiC show evidence for positive and negative cooperativity du...

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 proteinfolding rates.T he amino acid sequence of a protein and its chemical environment determine its native structure (1), but how this structure is determined from sequence remains one of the major unsolved problems in biology. The process of protein folding cannot be accomplished by random search through all possible conformations, because the number of structures available to an unfolded protein is too large; therefore there must be a biased or directional search in order for a protein to reach its native state. Small Two-State Proteins Show a Wide Range in Folding RatesThe simplest models for studying the protein-folding process are those that refold without intermediates (2). To date there are more than 20 examples of such simple systems. These proteins all fold cooperatively in a mono-exponential fashion from the denatured state, but the rates at which they fold span 6 orders of magnitude. What causes this remarkable variety of refolding rates? Folding Algorithms Based on the Topology of the Native State Have Predictive ValueRecently, several folding algorithms based on native structural information have been used to predict folding rates and nucleation sites (3-6). These efforts suggest that the topology of the final structure is an important determinant in the mechanism of protein folding.If native topology plays a major role in protein folding, is there a simple structural parameter that will capture this feature and explain the large range of observed folding rates? Recently, several simple parameters defining topological features of the native state have been shown to correlate well with proteinrefolding rates. The first and most commonly used parameter is relative contact order (RCO; ref. 7). Remarkably, RCO, which represents the normalized average sequence separation between contacting residues, was observed to correlate extremely well with the folding rates of a small set of two-state proteins. The lower the RCO, the faster the protein folds. Although it is possible to alter folding rates significantly through point mutations that do not change RCO, and there are proteins with similar RCOs and different folding rates (8), in general this correlation has improved as more two-state proteins have been characterized (9) and can explain the difference in folding rates among a family of structurally homologous pro...

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. Many proteins, however, 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 E. 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 and two-state RNases H, proteins with the same native state topology but altered regional stability, using a protein engineering approach. Our data suggest that 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.

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