The GroEL/GroES cis cavity as a passive anti-aggregation device.

The GroEL/GroES chaperonin folding chamber is an encapsulated space of approximately 65 A diameter with a hydrophilic wall, inside of which many cellular proteins reach the native state. The question of whether the cavity wall actively directs folding reactions or is playing a passive role has been open. We review past and recent observations and conclude that the chamber functions as a passive "Anfinsen cage" that prevents folding monomers from multimolecular aggregation.

Chaperonin chamber accelerates protein folding through passive action of preventing aggregation.

The original experiments reconstituting GroEL-GroES-mediated protein folding were carried out under "nonpermissive" conditions, where the chaperonin system was absolutely required and substrate proteins could not achieve the native state if diluted directly from denaturant into solution. Under "permissive" conditions, however, employing lower substrate concentration and lower temperature, some substrate proteins can be refolded both by the chaperonin system and while free in solution. For several of these, the protein refolds more rapidly inside the GroEL-GroES cis chamber than free in solution, suggesting that the chamber may have an active role in assisting protein folding. Here, we observe that the difference is caused by reversible multimolecular association while folding in solution, an avenue of kinetic partitioning that slows the overall rate of renaturation relative to the chaperonin chamber, where such associations cannot occur. For Rubisco, reversible aggregation during folding in solution was observed by gel filtration. For a mutant of maltose-binding protein (DM-MBP), the rate of folding in solution declined with increasing concentration, and the folding reaction produced light scattering. Under solution conditions where chloride was absent, however, light scattering no longer occurred, and DM-MBP folded at the same rate as in the cis cavity. In a further test, dihydrofolate reductase, thermally inactivated in the cis cavity or in solution, was substantially reactivated upon temperature downshift in the cis cavity but not in solution, where aggregation occurred. We conclude that the GroEL-GroES chamber behaves as a passive "Anfinsen cage" whose primary role is to prevent multimolecular association during folding.

Prion protein amyloid formation under native-like conditions involves refolding of the C-terminal alpha-helical domain.

Transmissible spongiform encephalopathies are associated with conformational conversion of the cellular prion protein, PrP(C), into a proteinase K-resistant, amyloid-like aggregate, PrP(Sc). Although the structure of PrP(Sc) remains enigmatic, recent studies have afforded increasingly detailed characterization of recombinant PrP amyloid. However, all previous studies were performed using amyloid fibrils formed in the presence of denaturing agents that significantly alter the folding state(s) of the precursor monomer. Here we report that PrP amyloid can also be generated under physiologically relevant conditions, where the monomeric protein is natively folded. Remarkably, site-directed spin labeling studies reveal that these fibrils possess a beta-core structure nearly indistinguishable from that of amyloid grown under denaturing conditions, where the C-terminal alpha-helical domain of the PrP monomer undergoes major refolding to a parallel and in-register beta-structure upon conversion. The structural similarity of fibrils formed under drastically different conditions strongly suggests that the common beta-sheet architecture within the approximately 160-220 core region represents a distinct global minimum in the PrP conversion free energy landscape. We also show that the N-terminal region of fibrillar PrP displays conformational plasticity, undergoing a reversible structural transition with an apparent pK(a) of approximately 5.3. The C-terminal region, on the other hand, retains its beta-structure over the pH range 1-11, whereas more alkaline buffer conditions denature the fibrils into constituent PrP monomers. This profile of pH-dependent stability is reminiscent of the behavior of brain-derived PrP(Sc), suggesting a substantial degree of structural similarity within the beta-core region of these PrP aggregates.

The emerging principles of mammalian prion propagation and transmissibility barriers: Insight from studies in vitro.

Self-perpetuating conformational conversion of the cellular prion protein PrP(C) into the beta-sheet-rich "scrapie" conformer (PrP(Sc)) is believed to be the central molecular event in pathogenesis of a group of diseases known as transmissible spongiform encephalopathies. Recent advances provide growing support for the notion that a misfolded protein alone might act as an infectious agent. Furthermore, findings regarding the mechanism of prion protein structural rearrangement, the role of folding intermediates in conformational conversion, and "conformational adaptability" in the propagation of prion amyloids in vitro yield molecular-level insight into such phenomena as inherited prion diseases, prion transmission barriers, and prion strains.

Early intermediate in human prion protein folding as evidenced by ultrarapid mixing experiments.

An important step toward understanding the mechanism of the PrP(C)-to-PrP(Sc) conversion is to elucidate the folding pathway(s) of the prion protein. On the basis of stopped-flow measurements, we recently proposed that the prion protein folds via a transient intermediate formed on the submillisecond time scale, and mutations linked to familial diseases result in a pronounced increase in the population of this intermediate. Here, we have extended these studies to continuous-flow measurements using a capillary mixing system with a time resolution of approximately 100 micros. This allowed us to directly observe two distinct phases in folding of the recombinant human prion protein 90-231, providing unambiguous evidence for rapid accumulation of an early intermediate (with a time constant of approximately 50 micros), followed by a rate-limiting folding step (with a time constant of approximately 700 micros). The present study also clearly demonstrates that the population of the intermediate is significantly increased at mildly acidic pH and in the presence of urea. A similar three-state folding behavior was observed for the Gerstmann-Straussler-Scheinker disease-associated F198S mutant, in which case the population of an intermediate was greatly increased as compared to that of the wild-type protein. Overall, the present data strongly suggest that this partially structured intermediate may be a direct monomeric precursor of the misfolded PrP(Sc) oligomer.

Polymorphism at residue 129 modulates the conformational conversion of the D178N variant of human prion protein 90-231.

One of the arguments in favor of the protein-only hypothesis of transmissible spongiform encephalopathies is the link between inherited prion diseases and specific mutations in the PRNP gene. One such mutation (Asp178 --> Asn) is associated with two distinct disorders: fatal familial insomnia or familial Creutzfeldt-Jakob disease, depending upon the presence of Met or Val at position 129, respectively. In this study, we have characterized the biophysical properties of recombinant human prion proteins (huPrP90-231) corresponding to the polymorphic variants D178N/M129 and D178N/V129. In comparison to the wild-type protein, both polymorphic forms of D178N huPrP show a greatly increased propensity for a conversion to beta-sheet-rich oligomers (at acidic pH) and thioflavine T-positive amyloid fibrils (at neutral pH). Importantly, the conversion propensity for the D178N variant is strongly dependent upon the M/V polymorphism at position 129, whereas under identical experimental conditions, no such dependence is observed for the wild-type protein. Amyloid fibrils formed by wild-type huPrP90-231 and the D178N variant are characterized by different secondary structures, and these structures are further modulated by residue 129 polymorphism. Although on the basis of only in vitro data, this study strongly suggests that polymorphism-dependent phenotypic variability of familial prion diseases may be linked to differences in biophysical properties of prion protein variants.

The effect of disease-associated mutations on the folding pathway of human prion protein.

Propagation of transmissible spongiform encephalopathies is believed to involve the conversion of cellular prion protein, PrP(C), into a misfolded oligomeric form, PrP(Sc). An important step toward understanding the mechanism of this conversion is to elucidate the folding pathway(s) of the prion protein. We reported recently (Apetri, A. C., and Surewicz, W. K. (2002) J. Biol. Chem. 277, 44589-44592) that the folding of wild-type prion protein can best be described by a three-state sequential model involving a partially folded intermediate. Here we have performed kinetic stopped-flow studies for a number of recombinant prion protein variants carrying mutations associated with familial forms of prion disease. Analysis of kinetic data clearly demonstrates the presence of partially structured intermediates on the refolding pathway of each PrP variant studied. In each case, the partially folded state is at least one order of magnitude more populated than the fully unfolded state. The present study also reveals that, for the majority of PrP variants tested, mutations linked to familial prion diseases result in a pronounced increase in the thermodynamic stability, and thus the population, of the folding intermediate. These data strongly suggest that partially structured intermediates of PrP may play a crucial role in prion protein conversion, serving as direct precursors of the pathogenic PrP(Sc) isoform.

Atypical effect of salts on the thermodynamic stability of human prion protein.

Prion diseases are associated with the conversion of cellular prion protein, PrPC, into a misfolded oligomeric form, PrPSc. Previous studies indicate that salts promote conformational conversion of the recombinant prion protein into a PrPSc-like form. To gain insight into the mechanism of this effect, here we have studied the influence of a number of salts (sodium sulfate, sodium fluoride, sodium acetate, and sodium chloride) on the thermodynamic stability of the recombinant human prion protein. Chemical unfolding studies in urea show that at low concentrations (below approximately 50 mm), all salts tested significantly reduced the thermodynamic stability of the protein. This highly unusual response to salts was observed for both the full-length prion protein as well as the N-truncated fragments huPrP90-231 and huPrP122-231. At higher salt concentrations, the destabilizing effect was gradually reversed, and salts behaved according to their ranking in the Hofmeister series. The present data indicate that electrostatic interactions play an unusually important role in the stability of the prion protein. The abnormal effect of salts is likely because of the ion-induced destabilization of salt bridges (Asp144-Arg148 and/or Asp147-Arg151) in the extremely hydrophilic helix 1. Contrary to previous suggestions, this effect is not due to the interaction of ions with the glycine-rich flexible N-terminal region of the prion protein. The results of this study suggest that ionic species present in the cellular environment may control the PrPC to PrPSc conversion by modulating the thermodynamic stability of the native PrPC isoform.

Kinetic intermediate in the folding of human prion protein.

Transmissible spongiform encephalopathies are associated with the conversion of cellular prion protein, PrP(C), into a misfolded oligomeric form, PrP(Sc). Here we have examined the kinetics of folding and unfolding reactions for the recombinant human prion protein C-terminal fragment 90-231 at pH 4.8 and 7.0. The stopped-flow data provide clear evidence for the population of an intermediate on the refolding pathway of the prion protein as indicated by a pronounced curvature in chevron plots and the presence of significant burst phase amplitude in the refolding kinetics. In addition to its role in the normal prion protein folding, this intermediate likely represents a crucial monomeric precursor of the pathogenic PrP(Sc) isoform.