The Nature of Proton Shuttling in Protic Ionic Liquid Fuel Cells

It has been proposed previously that protic ionic liquids (PILs) such as diethylmethylammonium triflate could be used as the electrolytes in nonhumidified, intermediate temperature H₂ fuel cells, potentially offering the prospect of high conductivity and performance, even under anhydrous conditions. In this contribution, a combination of electroanalytical chemistry and fuel‐cell polarization analyses is used to demonstrate for the first time that the pure PILs cannot support proton shuttling between the electrodes of fuel cells. Only through the inclusion of dissolved acidic or basic proton shuttles can viable protic ionic fuel cells be fabricated, which has major consequences for the use of these neoteric electrolytes in fuel cells.     

Order of steps in the cytochrome C folding pathway: evidence for a sequential stabilization mechanism

Previous work used hydrogen exchange (HX) experiments in kinetic and equilibrium modes to study the reversible unfolding and refolding of cytochrome c (Cyt c) under native conditions. Accumulated results now show that Cyt c is composed of five individually cooperative folding units, called foldons, which unfold and refold as concerted units in a stepwise pathway sequence. The first three steps of the folding pathway are linear and sequential. The ordering of the last two steps has been unclear because the fast HX of the amino acid residues in these foldons has made measurement difficult. New HX experiments done under slower exchange conditions show that the final two foldons do not unfold and refold in an obligatory sequence. They unfold separately and neither unfolding obligately contains the other, as indicated by their similar unfolding surface exposure and the specific effects of destabilizing and stabilizing mutations, pH change, and oxidation state. These results taken together support a sequential stabilization mechanism in which folding occurs in the native context with prior native-like structure serving to template the stepwise formation of subsequent native-like foldon units. Where the native structure of Cyt c requires sequential folding, in the first three steps, this is found. Where structural determination is ambiguous, in the final two steps, alternative parallel folding is found.     

Effects of Ligand Binding on the Mechanical Properties of Ankyrin Repeat Protein Gankyrin

Ankyrin repeat proteins are elastic materials that unfold and refold sequentially, repeat by repeat, under force. Herein we use atomistic molecular dynamics to compare the mechanical properties of the 7-ankyrin-repeat oncoprotein Gankyrin in isolation and in complex with its binding partner S6-C. We show that the bound S6-C greatly increases the resistance of Gankyrin to mechanical stress. The effect is specific to those repeats of Gankyrin directly in contact with S6-C, and the mechanical ‘hot spots’ of the interaction map to the same repeats as the thermodynamic hot spots. A consequence of stepwise nature of unfolding and the localized nature of ligand binding is that it impacts on all aspects of the protein''s mechanical behavior, including the order of repeat unfolding, the diversity of unfolding pathways accessed, the nature of partially unfolded intermediates, the forces required and the work transferred to the system to unfold the whole protein and its parts. Stepwise unfolding thus provides the means to buffer repeat proteins and their binding partners from mechanical stress in the cell. Our results illustrate how ligand binding can control the mechanical response of proteins. The data also point to a cellular mechano-switching mechanism whereby binding between two partner macromolecules is regulated by mechanical stress.     

The unfolding story of anthrax toxin translocation

The essential cellular functions of secretion and protein degradation require a molecular machine to unfold and translocate proteins either across a membrane or into a proteolytic complex. Protein translocation is also critical for microbial pathogenesis, namely bacteria can use translocase channels to deliver toxic proteins into a target cell. Anthrax toxin (Atx), a key virulence factor secreted by Bacillus anthracis, provides a robust biophysical model to characterize transmembrane protein translocation. Atx is comprised of three proteins: the translocase component, protective antigen (PA) and two enzyme components, lethal factor (LF) and oedema factor (OF). Atx forms an active holotoxin complex containing a ring-shaped PA oligomer bound to multiple copies of LF and OF. These complexes are endocytosed into mammalian host cells, where PA forms a protein-conducting translocase channel. The proton motive force unfolds and translocates LF and OF through the channel. Recent structure and function studies have shown that LF unfolds during translocation in a force-dependent manner via a series of metastable intermediates. Polypeptide-binding clamps located throughout the PA channel catalyse substrate unfolding and translocation by stabilizing unfolding intermediates through the formation of a series of interactions with various chemical groups and α-helical structure presented by the unfolding polypeptide during translocation.     

Association of partially folded lens betaB2-crystallins with the alpha-crystallin molecular chaperone

Age-related cataract is a result of crystallins, the predominant lens proteins, forming light-scattering aggregates. In the low protein turnover environment of the eye lens, the crystallins are susceptible to modifications that can reduce stability, increasing the probability of unfolding and aggregation events occurring. It is hypothesized that the alpha-crystallin molecular chaperone system recognizes and binds these proteins before they can form the light-scattering centres that result in cataract, thus maintaining the long-term transparency of the lens. In the present study, we investigated the unfolding and aggregation of (wild-type) human and calf betaB2-crystallins and the formation of a complex between alpha-crystallin and betaB2-crystallins under destabilizing conditions. Human and calf betaB2-crystallin unfold through a structurally similar pathway, but the increased stability of the C-terminal domain of human betaB2-crystallin relative to calf betaB2-crystallin results in the increased population of a partially folded intermediate during unfolding. This intermediate is aggregation-prone and prevents constructive refolding of human betaB2-crystallin, while calf betaB2-crystallin can refold with high efficiency. alpha-Crystallin can effectively chaperone both human and calf betaB2-crystallins from thermal aggregation, although chaperone-bound betaB2-crystallins are unable to refold once returned to native conditions. Ordered secondary structure is seen to increase in alpha-crystallin with elevated temperatures up to 60 degrees C; structure is rapidly lost at temperatures of 70 degrees C and above. Our experimental results combined with previously reported observations of alpha-crystallin quaternary structure have led us to propose a structural model of how activated alpha-crystallin chaperones unfolded betaB2-crystallin.     

Tracking unfolding and refolding of single GFPmut2 molecules

The unfolding and refolding kinetics of >600 single GFPmut2 molecules, entrapped in wet nanoporous silica gels, were followed by monitoring simultaneously the fluorescence emission of the anionic and neutral state of the chromophore, primed by two-photon excitation. The rate of unfolding, induced by guanidinium chloride, was determined by counting the number of single molecules that disappear in fluorescence images, under conditions that do not cause bleaching or photoinduced conversion between chromophore protonation states. The unfolding rate is of the order of 0.01 min(-1), and its dependence on denaturant concentration is very similar to that previously reported for high protein load gels. Upon rinsing the gels with denaturant-free buffer, the GFPmut2 molecules refold with rates >10 min(-1), with an apparently random distribution between neutral and anionic states, that can be very different from the preunfolding equilibrium. A subsequent very slow (lifetime of approximately 70 min) relaxation leads to the equilibrium distribution of the protonation states. This mechanism, involving one or more native-like refolding intermediates, is likely rate limited by conformational rearrangements that are undetectable in circular dichroism experiments. Several unfolding/refolding cycles can be followed on the same molecules, indicating full reversibility of the process and, noticeably, a bias of denaturated molecules toward refolding in the original protonation state.     

The sequence-dependent unfolding pathway plays a critical role in the amyloidogenicity of transthyretin

Human transthyretin (TTR) is an amyloidogenic protein whose aggregation is associated with several types of amyloid diseases. The following mechanism of TTR amyloid formation has been proposed. TTR tetramer at first dissociates into native monomers, which is the rate-limiting step in fibril formation. The monomeric species then partially unfold to form amyloidogenic intermediates that subsequently undergo a downhill self-assembly process. The amyloid deposit can be facilitated by disease-associated point mutations. However, only subtle structural differences were observed between the crystal structures of the wild type and the disease-associated variants. To investigate how single-point mutations influence the effective energy landscapes of TTR monomers, molecular dynamics (MD) simulations were performed on wild-type TTR and two pathogenic variants. Principal coordinate analysis on MD-generated ensembles has revealed multiple unfolding pathways for each protein. Amyloidogenic intermediates with the dislocated C strand-loop-D strand motif were observed only on the unfolding pathways of V30M and L55P variants and not for wild-type TTR. Our study suggests that the sequence-dependent unfolding pathway plays a crucial role in the amyloidogenicity of TTR. Analyses of side chain concerted motions indicate that pathogenic mutations on "edge strands" disrupt the delicate side chain correlated motions, which in turn may alter the sequence of unfolding events.     

Partially Folded Aggregation Intermediates of Human γD-, γC-, and γS-Crystallin Are Recognized and Bound by Human αB-Crystallin Chaperone

Human γ-crystallins are long-lived, unusually stable proteins of the eye lens exhibiting duplicated, double Greek key domains. The lens also contains high concentrations of the small heat shock chaperone α-crystallin, which suppresses aggregation of model substrates in vitro. Mature-onset cataract is believed to represent an aggregated state of partially unfolded and covalently damaged crystallins. Nonetheless, the lack of cell or tissue culture for anucleate lens fibers and the insoluble state of cataract proteins have made it difficult to identify the conformation of the human γ-crystallin substrate species recognized by human α-crystallin. The three major human lens monomeric γ-crystallins, γD, γC, and γS, all refold in vitro in the absence of chaperones, on dilution from denaturant into buffer. However, off-pathway aggregation of the partially folded intermediates competes with productive refolding. Incubation with human αB-crystallin chaperone during refolding suppressed the aggregation pathways of the three human γ-crystallin proteins. The chaperone did not dissociate or refold the aggregated chains under these conditions. The αB-crystallin oligomers formed long-lived stable complexes with their γD-crystallin substrates. Using α-crystallin chaperone variants lacking tryptophans, we obtained fluorescence spectra of the chaperone-substrate complex. Binding of substrate γ-crystallins with two or three of the four buried tryptophans replaced by phenylalanines showed that the bound substrate remained in a partially folded state with neither domain native-like. These in vitro results provide support for protein unfolding/protein aggregation models for cataract, with α-crystallin suppressing aggregation of damaged or unfolded proteins through early adulthood but becoming saturated with advancing age.     

Amyloidogenic unfolding intermediates differentiate sheep prion protein variants

Sheep is a unique example among mammalian species to present a strong correlation between genotype and prion disease susceptibility phenotype. Indeed a well-defined set of PrP polymorphisms at positions 136, 154 and 171 (sheep numbering) govern scrapie susceptibility, ranging from very high susceptibility for V136-R154-Q171 variant (VRQ) to resistance for A136-R154-R171 variant (ARR).To get better insight into the molecular mechanisms of scrapie susceptibility/resistance, the unfolding pathways of the different full-length recombinant sheep prion protein variants were analysed by differential scanning calorimetry in a wide range of pH. In the pH range 4.5-6.0, thermal unfolding occurs through a reversible one-step process while at pH <4.5 and >6.0 unfolding intermediates are formed, which are stable in the temperature range 65-80 degrees C. While these general behaviours are shared by all variants, VRQ and ARQ (susceptibility variants) show higher thermal stability than AHQ and ARR (resistance variants) and the formation of their unfolding intermediates requires higher activation energy than in the case of AHQ and ARR. Furthermore, secondary structures of the unfolding intermediates differentiate variants: ARR unfolding intermediate exhibits random coil structure, contrasting with the beta-sheet structure of VRQ and ARQ unfolding intermediates. The rate of the unfolding intermediate formation allows us to classify genetic variants along increasing scrapie susceptibility at pH 4.0, VRQ and ARQ rates being the highest. Rather poor correlation is observed at pH 7.2. Upon cooling, these intermediates refold into stable species, which are rich in beta-type secondary structures and, as revealed by thioflavin T fluorescence and electron microscopy, share amyloid characteristics. These results highlight the prion protein plasticity genetically modulated in sheep, and might provide a molecular basis for sheep predisposition to scrapie taking into account both thermodynamic stability and transconformation rate of prion protein.     

Folding kinetics and thermodynamics of Pseudomonas syringae effector protein AvrPto provide insight into translocation via the type III secretion system

In order to infect their hosts, many Gram-negative bacteria translocate agents of infection, called effector proteins, through the type III secretion system (TTSS) into the host cytoplasm. This process is thought to require at least partial unfolding of these agents, raising the question of how an effector protein might unfold to enable its translocation and then refold once it reaches the host cytoplasm. AvrPto is a well-studied effector protein of Pseudomonas syringae pv tomato. The presence of a readily observed unfolded population of AvrPto in aqueous solution and the lack of a known secretion chaperone make it ideal for studying the kinetic and thermodynamic characteristics that facilitate translocation. Application of Nzz exchange spectroscopy revealed a global, two-state folding equilibrium with 16% unfolded population, a folding rate of 1.8 s(-1), and an unfolding rate of 0.33 s(-1) at pH 6.1. TrAvrPto stability increases with increasing pH, with only 2% unfolded population observed at pH 7.0. The R(1) relaxation of TrAvrPto, which is sensitive to both the global anisotropy of folded TrAvrPto and slow exchange between folded and unfolded conformations, provided independent verification of the global kinetic rate constants. Given the acidic apoplast in which the pathogen resides and the more basic host cytoplasm, these results offer an intriguing mechanism by which the pH dependence of stability and slow folding kinetics of AvrPto would allow efficient translocation of the unfolded form through the TTSS and refolding into its functional folded form once inside the host.     

Differential scanning calorimetric,circular dichroism,and Fourier transform infrared spectroscopic characterization of the thermal unfolding of xylanase A from Streptomyces lividans

The thermal unfolding of xylanase A from Streptomyces lividans, and of its isolated substrate binding and catalytic domains, was studied by differential scanning calorimetry and Fourier transform infrared and circular dichroism spectroscopy. Our calorimetric studies show that the thermal denaturation of the intact enzyme is a complex process consisting of two endothermic events centered near 57 and 64 degrees C and an exothermic event centered near 75 degrees C, all of which overlap slightly on the temperature scale. A comparison of the data obtained with the intact enzyme and isolated substrate binding and catalytic domains indicate that the lower- and higher-temperature endothermic events are attributable to the thermal unfolding of the xylan binding and catalytic domains, respectively, whereas the higher-temperature exothermic event arises from the aggregation and precipitation of the denatured catalytic domain. Moreover, the thermal unfolding of the two domains of the native enzyme are thermodynamically independent and differentially sensitive to pH. The unfolding of the substrate binding domain is a reversible two-state process and, under appropriate conditions, the refolding of this domain to its native conformation can occur. In contrast, the unfolding of the catalytic domain is a more complex process in which two subdomains unfold independently over a similar temperature range. Also, the unfolding of the catalytic domain leads to aggregation and precipitation, which effectively precludes the refolding of the protein to its native conformation. These observations are compatible with the results of our spectroscopic studies, which show that the catalytic and substrate binding domains of the enzyme are structurally dissimilar and that their native conformations are unaffected by their association in the intact enzyme. Thus, the calorimetric and spectroscopic data demonstrate that the S. lividans xylanase A consists of structurally dissimilar catalytic and substrate binding domains that, although covalently linked, undergo essentially independent thermal denaturation. These observations provide valuable new insights into the structure and thermal stability of this enzyme and should assist our efforts at engineering xylanases that are more thermally robust and otherwise better suited for industrial applications.     

Role of conformational dynamics in pathogenic protein aggregation

The accumulation of pathogenic protein oligomers and aggregates is associated with several devastating amyloid diseases. As protein aggregation is a multi-step nucleation-dependent process beginning with unfolding or misfolding of the native state, it is important to understand how innate protein dynamics influence aggregation propensity. Kinetic intermediates composed of heterogeneous ensembles of oligomers are frequently formed on the aggregation pathway. Characterization of the structure and dynamics of these intermediates is critical to the understanding of amyloid diseases since oligomers appear to be the main cytotoxic agents. In this review, we highlight recent biophysical studies of the roles of protein dynamics in driving pathogenic protein aggregation, yielding new mechanistic insights that can be leveraged for design of aggregation inhibitors.     

Conformational prerequisites for formation of amyloid fibrils from histones

We demonstrate that bovine core histones are natively unfolded proteins in solutions with low ionic strength due to their high net positive charge at pH 7.5. Using a variety of biophysical techniques we characterized their conformation as a function of pH and ionic strength, as well as correlating the conformation with aggregation and amyloid fibril formation. Tertiary structure was absent under all conditions except at pH 7.5 and high ionic strength. The addition of trifluoroethanol or high ionic strength induced significant alpha-helical secondary structure at pH 7.5. At low pH and high salt concentration, small-angle X-ray scattering and SEC HPLC indicate the histones are present as a hexadecamer of globular subunits. The secondary structure at low pH was independent of the ionic strength or presence of TFE, as judged by FTIR. The data indicate that histones are able to adopt five different relatively stable conformations; this conformational variability probably reflects, in part, their intrinsically disordered structure. Under most of the conditions studied the histones formed amyloid fibrils with typical morphology as seen by electron microscopy. In contrast to most aggregation/amyloidogenic systems, the kinetics of fibrillation showed an inverse dependence on histone concentration; we attribute this to partitioning to a faster pathway leading to non-fibrillar self-associated aggregates at higher protein concentrations. The rate of fibril formation was maximal at low pH, and decreased to zero by pH 10. The kinetics of fibrillation were very dependent on the ionic strength, increasing with increasing salt concentration, and showing marked dependence on the nature of the ions; interestingly Gdn.HCl increased the rate of fibrillation, although much less than NaCl. Different ions also differentially affected the rate of nucleation and the rate of fibril elongation.     

Measuring “Unmeasurable” Folding Kinetics of Proteins by Single-Molecule Force Spectroscopy

Folding and unfolding are fundamental biological processes in cell and are important for the biological functions of proteins. Characterizing the folding and unfolding kinetics of proteins is important for understanding the energetic landscape leading to the active native conformations of these molecules. However, the thermal or chemical-induced unfolding of many proteins is irreversible in vitro, precluding characterization of the folding kinetics of such proteins, just as it is impossible to “un-boil” an egg. Irreversible unfolding often manifests as irreversible aggregation of unfolded polypeptide chains, which typically occurs between denatured protein molecules in response to the exposure of hydrophobic residues to solvent. An example of such a protein where thermal denaturation results in irreversible aggregation is the β-1,4 endoxylanase from Bacillus circulans (BCX). Here, we report the use of single-molecule atomic force microscopy to directly measure the folding kinetics of BCX in vitro. By mechanically unfolding BCX, we essentially allowed only one unfolded molecule to exist in solution at a given time, effectively eliminating the possibility for aggregation. We found that BCX can readily refold back to the native state, allowing us to measure its folding kinetics for the first time. Our results demonstrate that single-molecule force-spectroscopy-based methods can adequately tackle the challenge of “un-boiling eggs”, providing a general methodology to characterize the folding kinetics of many proteins that suffer from irreversible denaturation and thus cannot be characterized using traditional equilibrium methodologies.     

Role of aggregation conditions in structure, stability, and toxicity of intermediates in the Abeta fibril formation pathway

beta-amyloid peptide (Abeta) is one of the main protein components of senile plaques associated with Alzheimer's disease (AD). Abeta readily aggregates to forms fibrils and other aggregated species that have been shown to be toxic in a number of studies. In particular, soluble oligomeric forms are closely related to neurotoxicity. However, the relationship between neurotoxicity and the size of Abeta aggregates or oligomers is still under investigation. In this article, we show that different Abeta incubation conditions in vitro can affect the rate of Abeta fibril formation, the conformation and stability of intermediates in the aggregation pathway, and toxicity of aggregated species formed. When gently agitated, Abeta aggregates faster than Abeta prepared under quiescent conditions, forming fibrils. The morphology of fibrils formed at the end of aggregation with or without agitation, as observed in electron micrographs, is somewhat different. Interestingly, intermediates or oligomers formed during Abeta aggregation differ greatly under agitated and quiescent conditions. Unfolding studies in guanidine hydrochloride indicate that fibrils formed under quiescent conditions are more stable to unfolding in detergent than aggregation associated oligomers or Abeta fibrils formed with agitation. In addition, Abeta fibrils formed under quiescent conditions were less toxic to differentiated SH-SY5Y cells than the Abeta aggregation associated oligomers or fibrils formed with agitation. These results highlight differences between Abeta aggregation intermediates formed under different conditions and provide insight into the structure and stability of toxic Abeta oligomers.     

Change of thermal stability of colicin E7 triggered by acidic pH suggests the existence of unfolded intermediate during the membrane-translocation phase

Purified colicin E7 was analyzed by CD spectrum and gel filtration chromatography in a mimicking membrane-translocation phase. It was found that the CD spectra of colicin E7 at pH 7 and pH 2.5 were similar. Although the melting temperature of the protein shifted from 54.5°C to 34°C at low pH, the thermal denaturation curves of colicin E7 at different pH conditions still fit a two-state model. These experimental results imply that a minor structural change, triggered by acidic pH, for instance, may reduce the energy required for protein melting. In contrast to the minor change in secondary structure at different pH conditions, we observed that, in vitro, all monomeric colicin E7s converted into multimer-like conformations after recovering from the partial unfolding process. This multimeric form of colicin can only be dissociated by formamide and guanidine hydrochloride, indicating that this protein complex is indeed formed by aggregation of the monomeric colicins. Most interestingly, the aggregated colicins still perform in vivo bacteriocidal activity. We suggest that in a partial unfolding state the colicin is prepared for binding to the specific targets for translocation through the membrane. However, in the absence of specific targets in vitro these unfold intermediates may therefore aggregate into the multimeric form of colicins. Proteins 32:17–25, 1998. © 1998 Wiley-Liss, Inc.     

Equilibrium unfolding of kinetically stable serine protease milin: the presence of various active and inactive dimeric intermediates

Kinetically stable homodimeric serine protease milin reveals high conformational stability against temperature, pH and chaotrope [urea, guanidine hydrochloride (GuHCl) and guanidine isothiocynate (GuSCN)] denaturation as probed by circular dichroism, fluorescence, differential scanning calorimetry and activity measurements. GuSCN induces complete unfolding in milin, whereas temperature, urea and GuHCl induce only partial unfolding even at low pH, through several intermediates with distinct characteristics. Some of these intermediates are partially active (viz. in urea and 2 M GuHCl at pH 7.0), and some exhibited strong ANS binding as well. All three tryptophans in the protein seem to be buried in a rigid, compact core as evident from intrinsic fluorescence measurements coupled to equilibrium unfolding experiments. The protein unfolds as a dimer, where the unfolding event precedes dimer dissociation as confirmed by hydrodynamic studies. The solution studies performed here along with previous biochemical characterization indicate that the protein has α-helix and β-sheet rich regions or structural domains that unfold independently, and the monomer association is isologous. The complex unfolding pathway of milin and the intermediates has been characterized. The physical, physiological and probable therapeutic importance of the results has been discussed.     

Multiple unfolding intermediates of human placental alkaline phosphatase in equilibrium urea denaturation.

Alkaline phosphatase is an enzyme with a typical alpha/beta hydrolase fold. The conformational stability of the human placental alkaline phosphatase was examined with the chemical denaturant urea. The red shifts of fluorescence spectra show a complex unfolding process involving multiple equilibrium intermediates indicating differential stability of the subdomains of the enzyme. None of these unfolding intermediates were observed in the presence of 83 mM NaCl, indicating the importance of ionic interactions in the stabilization of the unfolding intermediates. Guanidinium chloride, on the other hand, could stabilize one of the unfolding intermediates, which is not a salt effect. Some of the unfolding intermediates were also observed in circular dichroism spectroscopy, which clearly indicates steady loss of helical structure during unfolding, but very little change was observed for the beta strand content until the late stage of the unfolding process. The enzyme does not lose its phosphate-binding ability after substantial tertiary structure changes, suggesting that the substrate-binding region is more resistant to chemical denaturant than the other structural domains. Global analysis of the fluorescence spectral change demonstrated the following folding-unfolding process of the enzyme: N <--> I(1) <--> I(2) <--> I(3) <--> I(4) <--> I(5) <--> D. These discrete intermediates are stable at urea concentrations of 2.6, 4.1, 4.7, 5.5, 6.6, and 7.7 M, respectively. These intermediates are further characterized by acrylamide and/or potassium iodide quenching of the intrinsic fluorescence of the enzyme and by the hydrophobic probes, 1-anilinonaphthalene-8-sulfonic acid and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid. The stepwise unfolding process was interpreted by the folding energy landscape in terms of the unique structure of the enzyme. The rigid central beta-strand domain is surrounded by the peripheral alpha-helical and coil structures, which are marginally stable toward a chemical denaturant.     

Stepwise unfolding of a β barrel protein by the AAA+ ClpXP protease

In the AAA+ ClpXP protease, ClpX uses the energy of ATP binding and hydrolysis to unfold proteins before translocating them into ClpP for degradation. For proteins with C-terminal ssrA tags, ClpXP pulls on the tag to initiate unfolding and subsequent degradation. Here, we demonstrate that an initial step in ClpXP unfolding of the 11-stranded β barrel of superfolder GFP-ssrA involves extraction of the C-terminal β strand. The resulting 10-stranded intermediate is populated at low ATP concentrations, which stall ClpXP unfolding, and at high ATP concentrations, which support robust degradation. To determine if stable unfolding intermediates cause low-ATP stalling, we designed and characterized circularly permuted GFP variants. Notably, stalling was observed for a variant that formed a stable 10-stranded intermediate but not for one in which this intermediate was unstable. A stepwise degradation model in which the rates of terminal-strand extraction, strand refolding or recapture, and unfolding of the 10-stranded intermediate all depend on the rate of ATP hydrolysis by ClpXP accounts for the observed changes in degradation kinetics over a broad range of ATP concentrations. Our results suggest that the presence or absence of unfolding intermediates will play important roles in determining whether forced enzymatic unfolding requires a minimum rate of ATP hydrolysis.     

The role of heat-shock and chaperone proteins in protein folding: possible molecular mechanisms.

Recently some heat-shock proteins have been linked to functions of 'chaperoning' protein folding in vivo. Here current experimental evidence is reviewed and possible requirements for such an activity are discussed. It is proposed that one mode of chaperone action is to actively unfold misfolded or badly aggregated proteins to a conformation from which they could refold spontaneously; that improperly folded proteins are recognized by excessive stretches of solvent-exposed backbone, rather than by exposed hydrophobic patches; and that the molecular mechanism for unfolding is either repeated binding and dissociation ('plucking') or translocation of the protein backbone through a binding cleft ('threading'), allowing the threaded chain to refold spontaneously. The observed hydrolysis of ATP would provide the energy for active unfolding. These hypotheses can be applied to both monomeric folding and oligomeric assembly and are sufficiently detailed to be open to directed experimental verification.