Off-pathway State Research Articles

β-Barrels and Amyloids: Structural Transitions, Biological Functions, and Pathogenesis.

Insoluble protein aggregates with fibrillar morphology called amyloids and β-barrel proteins both share a β-sheet-rich structure. Correctly folded β-barrel proteins can not only function in monomeric (dimeric) form, but also tend to interact with one another—followed, in several cases, by formation of higher order oligomers or even aggregates. In recent years, findings proving that β-barrel proteins can adopt cross-β amyloid folds have emerged. Different β-barrel proteins were shown to form amyloid fibrils in vitro. The formation of functional amyloids in vivo by β-barrel proteins for which the amyloid state is native was also discovered. In particular, several prokaryotic and eukaryotic proteins with β-barrel domains were demonstrated to form amyloids in vivo, where they participate in interspecies interactions and nutrient storage, respectively. According to recent observations, despite the variety of primary structures of amyloid-forming proteins, most of them can adopt a conformational state with the β-barrel topology. This state can be intermediate on the pathway of fibrillogenesis (“on-pathway state”), or can be formed as a result of an alternative assembly of partially unfolded monomers (“off-pathway state”). The β-barrel oligomers formed by amyloid proteins possess toxicity, and are likely to be involved in the development of amyloidoses, thus representing promising targets for potential therapy of these incurable diseases. Considering rapidly growing discoveries of the amyloid-forming β-barrels, we may suggest that their real number and diversity of functions are significantly higher than identified to date, and represent only “the tip of the iceberg”. Here, we summarize the data on the amyloid-forming β-barrel proteins, their physicochemical properties, and their biological functions, and discuss probable means and consequences of the amyloidogenesis of these proteins, along with structural relationships between these two widespread types of β-folds.

Split conformation of Chaetomium thermophilum Hsp104 disaggregase

Hsp104 and its bacterial homolog ClpB form hexameric ring structures and mediate protein disaggregation. The disaggregated polypeptide is thought to thread through the central channel of the ring. However, the dynamic behavior of Hsp104 during disaggregation remains unclear. Here, we reported the stochastic conformational dynamics and a split conformation of Hsp104 disaggregase from Chaetomium thermophilum (CtHsp104) in the presence of ADP by X-ray crystallography, cryo-electron microscopy (EM), and high-speed atomic force microscopy (AFM). ADP-bound CtHsp104 assembles into a 65 left-handed spiral filament in the crystal structure at a resolution of 2.7Å. The unit of the filament is a hexamer of the split spiral structure. In the cryo-EM images, staggered and split hexameric rings were observed. Further, high-speed AFM observations showed that a substrate addition enhanced the conformational change and increased the split structure's frequency. Our data suggest that split conformation is an off-pathway state of CtHsp104 during disaggregation.

Investigation of West Nile Virus Off-Pathway Membrane Fusion Mechanisms using Kinetic Modeling

West Nile virus (WNV) is a positive-sense, single-stranded RNA virus that causes febrile illness (West Nile fever) in humans. It is a member of the flavivirus genus, which includes dengue virus, tick-borne encephalitis, yellow fever virus, and Zika virus. To infect a host cell, West Nile virions first bind to the host cell plasma membrane. Upon binding, the virions are taken up via endocytosis, during which a decrease in pH causes the virions to fuse with the endosomal membrane. Fusion is mediated by the viral glycoprotein E, which is triggered by endosomal pH and undergoes a dimer-to-trimer rearrangement during the fusion process. Previously, we and others used simple chemical kinetics modeling and a cellular automaton simulation to show that an off-pathway state is necessary to fit WNV and Zika virus fusion data. Here, we investigate hypotheses to further explore what the off-pathway state entails, including whether the off-pathway state occurs before or after fusion loop insertion, and whether it occurs as a monomer or a multimer state. We also discuss comparisons between the ability of the off-pathway model to fit both West Nile virus and dengue virus fusion data.

Kinetic Modeling of West Nile Virus Fusion Indicates an Off-Pathway State.

West Nile virus (WNV) is a prominent mosquito-borne flavivirus that causes febrile illness in humans. To infect host cells, WNV virions first bind to plasma membrane receptors, then initiate membrane fusion following endocytosis. The viral transmembrane E protein, triggered by endosomal pH, catalyzes fusion while undergoing a dimer-to-trimer transition. Previously, single-particle WNV fusion data was interrogated with a stochastic cellular automaton simulation, which modeled the E proteins during the fusion process. The results supported a linear fusion mechanism, with E protein trimerization being rate-limiting. Here, we present corrections to the previous simulation, and apply them to the WNV fusion data. We observe that a linear mechanism is no longer sufficient to fit the data. Instead, an off-pathway state is necessary; these results are corroborated by per virus chemical kinetics modeling. When compared with a similar Zika virus fusion model, this suggests that off-pathway fusion mechanisms may characterize flaviviruses more broadly.

Unraveling mechanisms from waiting time distributions in single-nanoparticle catalysis

The catalytic conversion of substrates to products at the surface of a single nanoparticle cluster can now be resolved at the molecular scale and the waiting time between individual product turnovers measured with precision. The distribution of waiting times and, in particular, their means and variances can thus be obtained experimentally. Here, we show how theoretical modeling based on the chemical master equation (CME) provides a powerful tool to extract catalytic mechanisms and rate parameters from such experimental data. Conjecturing a family of mechanisms that both include and exclude surface restructuring, we obtain the mean and variance of their waiting times from the CME. A detailed analysis of the link between mechanism topology and waiting time dispersion, then, allows us to select several candidate mechanisms, with branched topologies, that can reproduce experimental data. From these, the least complex model that best matches experimental data is chosen as the minimum model. The CME modeling extracts the Langmuir-Hinshelwood mechanism for product formation and two-pathway mechanism for product dissociation, with substantial off-pathway state fluctuations due to surface restructuring dynamics, as the minimal model consistent with data. Our work, thus, provides a mechanistic origin of the coupling between the kinetics of catalytic turnovers and surface restructuring dynamics and yields a systematic way to compute catalytic rates from distributions of waiting times between product turnovers in the presence of surface restructuring.

The elemental mechanism of transcriptional pausing.

Transcriptional pausing underlies regulation of cellular RNA biogenesis. A consensus pause sequence that acts on RNA polymerases (RNAPs) from bacteria to mammals halts RNAP in an elemental paused state from which longer-lived pauses can arise. Although the structural foundations of pauses prolonged by backtracking or nascent RNA hairpins are recognized, the fundamental mechanism of the elemental pause is less well-defined. Here we report a mechanistic dissection that establishes the elemental pause signal (i) is multipartite; (ii) causes a modest conformational shift that puts γ-proteobacterial RNAP in an off-pathway state in which template base loading but not RNA translocation is inhibited; and (iii) allows RNAP to enter pretranslocated and one-base-pair backtracked states easily even though the half-translocated state observed in paused cryo-EM structures rate-limits pause escape. Our findings provide a mechanistic basis for the elemental pause and a framework to understand how pausing is modulated by sequence, cellular conditions, and regulators.

PH Dependence of Zika Membrane Fusion Kinetics Reveals an Off-Pathway State

The recent spread of Zika virus stimulated extensive research on its structure, pathogenesis, and immunology, but mechanistic study of entry has lagged behind, in part due to the lack of a defined reconstituted system. Here, we report Zika membrane fusion measured using a platform that bypasses these barriers, enabling observation of single-virus fusion kinetics without receptor reconstitution. Surprisingly, target membrane binding and low pH are sufficient to trigger viral hemifusion to liposomes containing only neutral lipids. Second, although the extent of hemifusion strongly depends on pH, hemifusion rates are relatively insensitive to pH. Kinetic analysis shows that an off-pathway state is required to capture this pH-dependence and suggests this may be related to viral inactivation. Our surrogate-receptor approach thus yields new understanding of flaviviral entry mechanisms and should be applicable to many emerging viruses.

FRET studies of various conformational states adopted by transthyretin.

Transthyretin (TTR) is an extracellular protein able to deposit into well-defined protein aggregates called amyloid, in pathological conditions known as senile systemic amyloidosis, familial amyloid polyneuropathy, familial amyloid cardiomyopathy and leptomeningeal amyloidosis. At least three distinct partially folded states have been described for TTR, including the widely studied amyloidogenic state at mildly acidic pH. Here, we have used fluorescence resonance energy transfer (FRET) experiments in a monomeric variant of TTR (M-TTR) and in its W41F and W79F mutants, taking advantage of the presence of a unique, solvent-exposed, cysteine residue at position 10, that we have labelled with a coumarin derivative (DACM, acceptor), and of the two natural tryptophan residues at positions 41 and 79 (donors). Trp41 is located in an ideal position as it is one of the residues of β-strand C, whose degree of unfolding is debated. We found that the amyloidogenic state at low pH has the same FRET efficiency as the folded state at neutral pH in both M-TTR and W79F-M-TTR, indicating an unmodified Cys10-Trp41 distance. The partially folded state populated at low denaturant concentrations also has a similar FRET efficiency, but other spectroscopic probes indicate that it is distinct from the amyloidogenic state at acidic pH. By contrast, the off-pathway state accumulating transiently during refolding has a higher FRET efficiency, indicating non-native interactions that reduce the Cys10-Trp41 spatial distance, revealing a third distinct conformational state. Overall, our results clarify a negligible degree of unfolding of β-strand C in the formation of the amyloidogenic state and establish the concept that TTR is a highly plastic protein able to populate at least three distinct conformational states.

Oncogenic Mutations Differentially Affect Bax Monomer, Dimer, and Oligomeric Pore Formation in the Membrane.

Dysfunction of Bax, a pro-apoptotic regulator of cellular metabolism is implicated in neurodegenerative diseases and cancer. We have constructed the first atomistic models of the Bax oligomeric pore consisting with experimental residue-residue distances. The models are stable, capturing well double electron-electron resonance (DEER) spectroscopy measurements and provide structural details in line with the DEER data. Comparison with the latest experimental results revealed that our models agree well with both Bax and Bak pores, pointed to a converged structural arrangement for Bax and Bak pore formation. Using multi-scale molecular dynamics simulations, we probed mutational effects on Bax transformation from monomer → dimer → membrane pore formation at atomic resolution. We observe that two cancer-related mutations, G40E and S118I, allosterically destabilize the monomer and stabilize an off-pathway swapped dimer, preventing productive pore formation. This observation suggests a mechanism whereby the mutations may work mainly by over-stabilizing the monomer → dimer transformation toward an unproductive off-pathway swapped-dimer state. Our observations point to misfolded Bax states, shedding light on the molecular mechanism of Bax mutation-elicited cancer. Most importantly, the structure of the Bax pore facilitates future study of releases cytochrome C in atomic detail.

Ionic Strength Modulation of the Free Energy Landscape of Aβ40 Peptide Fibril Formation.

Protein misfolding and formation of cross-β structured amyloid fibrils are linked to many neurodegenerative disorders. Although recently developed quantitative approaches have started to reveal the molecular nature of self-assembly and fibril formation of proteins and peptides, it is yet unclear how these self-organization events are precisely modulated by microenvironmental factors, which are known to strongly affect the macroscopic aggregation properties. Here, we characterize the explicit effect of ionic strength on the microscopic aggregation rates of amyloid β peptide (Aβ40) self-association, implicated in Alzheimer's disease. We found that physiological ionic strength accelerates Aβ40 aggregation kinetics by promoting surface-catalyzed secondary nucleation reactions. This promoted catalytic effect can be assigned to shielding of electrostatic repulsion between monomers on the fibril surface or between the fibril surface itself and monomeric peptides. Furthermore, we observe the formation of two different β-structured states with similar but distinct spectroscopic features, which can be assigned to an off-pathway immature state (Fβ*) and a mature stable state (Fβ), where salt favors formation of the Fβ fibril morphology. Addition of salt to preformed Fβ* accelerates transition to Fβ, underlining the dynamic nature of Aβ40 fibrils in solution. On the basis of these results we suggest a model where salt decreases the free-energy barrier for Aβ40 folding to the Fβ state, favoring the buildup of the mature fibril morphology while omitting competing, energetically less favorable structural states.

Pausing in Escherichia coli Transcription Initiation

An essential and highly regulated step in gene expression is transcription initiation. After promoter binding and DNA unwinding (‘bubble opening’) and in the presence of nucleoside triphosphates (NTPs), the RNA polymerase (RNAP)-promoter initial transcribing complex (RPitc) engages in ‘abortive initiation’, a process in which RNAP cycles between synthesis and release of short RNA transcripts. In abortive initiation, RPitc is believed to undergo a sequence of transitions between different initiation sub-states. The kinetics of the production of a full RNA transcript starting at a late initiation sub-state is expected to be similar or faster than the kinetics measured from an earlier initiation sub-state. To test this hypothesis, we developed a novel in vitro single-run quenched kinetics transcription assay based on the detection and quantification of run-off transcripts. Using this assay and corroborating it with gel-based and magnetic tweezer assays, testing two different promoters, we surprisingly found that run-off transcription kinetics starting from late initiation sub-states is slower than kinetics starting from earlier initiation sub-states. When the same kinetic measurements were performed in the presence of the transcription elongation factor GreA, the kinetics starting from a late initiation sub-state was accelerated. Experimental results suggest that as a function of shortage in NTPs RPitc can enter an off-pathway state in which the nascent RNA is in a backtracked, paused position, awaiting incorporation of the missing NTP. Our findings suggest that pausing at distinct stages of transcription initiation could regulate gene expression under stressed conditions.

Impact of Alkyl Spacer Length on Aggregation Pathways in Kinetically Controlled Supramolecular Polymerization.

We have investigated the kinetic and thermodynamic supramolecular polymerizations of a series of amide-functionalized perylene bisimide (PBI) organogelator molecules bearing alkyl spacers of varied lengths (ethylene to pentylene chains, PBI-1-C2 to PBI-1-C5) between the amide and PBI imide groups. These amide-functionalized PBIs form one-dimensional fibrous nanostructures as the thermodynamically favored states in solvents of low polarity. Our in-depth studies revealed, however, that the kinetic behavior of their supramolecular polymerization is dependent on the spacer length. Propylene- and pentylene-tethered PBIs follow a similar polymerization process as previously observed for the ethylene-tethered PBI. Thus, the monomers of these PBIs are kinetically trapped in conformationally restricted states through intramolecular hydrogen bonding between the amide and imide groups. In contrast, the intramolecularly hydrogen-bonded monomers of butylene-tethered PBI spontaneously self-assemble into nanoparticles, which constitute an off-pathway aggregate state with regard to the thermodynamically stable fibrous supramolecular polymers obtained. Thus, for this class of π-conjugated system, an unprecedented off-pathway aggregate with high kinetic stability could be realized for the first time by introducing an alkyl linker of optimum length (C4 chain) between the amide and imide groups. Our current system with an energy landscape of two competing nucleated aggregation pathways is applicable to the kinetic control over the supramolecular polymerization by the seeding approach.

The Atomistic Mechanism of Conformational Transition of Adenylate Kinase Investigated by Lorentzian Structure-Based Potential.

We present a new all-atom structure-based method to study protein conformational transitions using Lorentzian attractive interactions based on native structures. The variability of each native contact is estimated based on evolutionary information using a machine learning method. To test the validity of this approach, we have investigated the conformational transition of adenylate kinase (ADK). The intrinsic boundedness of the Lorentzian attractive interactions facilitated frequent conformational transitions, and consequently we were able to observe more than 1000 structural interconversions between the open and closed states of ADK out of a total of 6 μs MD simulations. ADK has three domains: the nucleoside monophosphate (NMP) binding domain, the LID-domain, and the CORE domain, which catalyze the interconversion between ATP and ADP. We identified two transition states: a more frequent LID-closed-NMP-open (TS1) state and a less frequent LID-open-NMP-closed (TS2) state. The transition was found to be symmetric in both directions via TS1. We also obtained an off-pathway metastable state that was previously observed with physics-based all-atom simulations but not with coarse-grained models. In the metastable state, the LID domain was slightly twisted and formed contacts with the NMP domain. Our model correctly identified a total of 14 out of the top 16 residues with highest fluctuation by NMR experiment, thus showing excellent agreement with experimental NMR relaxation data and overwhelmingly better results than existing models.

A radical intermediate in the conversion of pentachlorophenol to tetrachlorohydroquinone by Sphingobium chlorophenolicum.

Pentachlorophenol (PCP) hydroxylase, the first enzyme in the pathway for degradation of PCP in Sphingobium chlorophenolicum, is an unusually slow flavin-dependent monooxygenase (kcat = 0.02 s–1) that converts PCP to a highly reactive product, tetrachlorobenzoquinone (TCBQ). Using stopped-flow spectroscopy, we have shown that the steps up to and including formation of TCBQ are rapid (5–30 s–1). Before products can be released from the active site, the strongly oxidizing TCBQ abstracts an electron from a donor at the active site, possibly a cysteine residue, resulting in an off-pathway diradical state that only slowly reverts to an intermediate capable of completing the catalytic cycle. TCBQ reductase, the second enzyme in the PCP degradation pathway, rescues this nonproductive complex via two fast sequential one-electron transfers. These studies demonstrate how adoption of an ancestral catalytic strategy for conversion of a substrate with different steric and electronic properties can lead to subtle yet (literally) radical changes in enzymatic reaction mechanisms.

Distinct Effects of Guanidine Thiocyanate on the Structure of Superfolder GFP

Having a high folding efficiency and a low tendency to aggregate, the superfolder GFP (sfGFP) offers a unique opportunity to study the folding of proteins that have a β-barrel topology. Here, we studied the unfolding–refolding of sfGFP that was induced by guanidine thiocyanate (GTC), which is a stronger denaturing agent than GdnHCl or urea. Structural perturbations of sfGFP were studied by spectroscopic methods (absorbance, fluorescence, and circular dichroism), by acrylamide quenching of tryptophan and green chromophore fluorescence, and by size-exclusion chromatography. Low concentrations of GTC (up to 0.1 M) induce subtle changes in the sfGFP structure. The pronounced changes in the visible absorption spectrum of sfGFP which are accompanied by a dramatic decrease in tryptophan and green chromophore fluorescence was recorded in the range 0–0.7 M GNC. These alterations of sfGFP characteristics that erroneously can be mixed up with appearance of intermediate state in fact have pure spectroscopic but not structural nature. Higher concentrations of GTC (from 0.7 to 1.7 M), induce a disruption of the sfGFP structure, that is manifested in simultaneous changes of all of the detected parameters. Full recovery of native properties of denaturated sfGFP was observed after denaturant removal. The refolding of sfGFP passes through the accumulation of the off-pathway intermediate state, in which inner alpha-helix and hence green chromophore and Trp57 are still not tuned up to (properly integrated into) the already formed β-barrel scaffold of protein. Incorporation of the chromophore in the β-barrel in the pathway of refolding and restoration of its ability to fluoresce occur in a narrow range of GTC concentrations from 1.0 to 0.7 M, and a correct insertion of Trp 57 occurs at concentrations ranging from 0.7 to 0 M GTC. These two processes determine the hysteresis of protein unfolding and refolding.

The mechanism of transcription antitermination by RfaH

RfaH is a bacterial antitermination factor whose function depends on a 12‐nt ops element located in the leader regions of several operons; the ops element both induces RNA polymerase (RNAP) pausing and serves as a recognition site for RfaH. During recruitment, RfaH establishes transient contacts with the ops bases in the non‐template DNA strand exposed on the RNAP surface. After recruitment, RfaH remains bound to RNAP throughout transcription of the entire operon and switches RNAP into a processive state using four mechanisms. First, RfaH competes with σ and prevents σ‐dependent arrest during elongation. Second, RfaH competes with NusG and thus indirectly reduces Rho‐dependent termination. Third, RfaH induces forward translocation of RNAP, thereby inhibiting isomerization into an off‐pathway state that occurs from a pre‐translocated conformation. Fourth, RfaH establishes simultaneous contacts with the β’ clamp and the β lobe, the two elements that maintain the RNAP grip on the nucleic acid chains, thereby preventing the clamp opening that is thought to occur during pausing. While the first three mechanisms are specific to the RfaH‐like proteins, the last mechanism is likely broadly conserved. We propose that structurally diverse antiterminators from bacteria to humans solidify the transcription elongation complex to inhibit conformational transitions that underlie pausing and termination.

The mechanism of amyloid-fibril formation by stefin B: temperature and protein concentration dependence of the rates.

Cystatins, a family of structurally related cysteine proteinase inhibitors, have proved to be useful model system to study amyloidogenesis. We have extended previous studies of the kinetics of amyloid-fibril formation by human stefin B (cystatin B) and some of its mutants, and proposed an improved model for the reaction. Overall, the observed kinetics follow the nucleation and growth behavior observed for many other amyloidogenic proteins. The minimal kinetic scheme that best fits measurements of changes in CD and thioflavin T fluorescence as a function of protein concentration and temperature includes nucleation (modeled as N(I) irreversible transitions with equivalent rates (k(I)), which fitted with N(I) = 64), fibril growth and nonproductive oligomerization, best explained by an off-pathway state with a rate-limiting escape rate. Three energies of activation were derived from global fitting to the minimal kinetic scheme, and independently through the fitting of the individual component rates. Nucleation was found to be a first-order process within an oligomeric species with an enthalpy of activation of 55 +/- 4 kcal mol(-1). Fibril growth was a second-order process with an enthalpy of activation (27 +/- 5 kcal mol(-1)), which is indistinguishable from that of tetramer formation by cystatins, which involves limited conformational changes including proline trans to cis isomerization. The highest enthalpy of activation (95 +/- 5 kcal mol(-1) at 35 degrees C), characteristic of a substantial degree of unfolding as observed prior to domain-swapping reactions, equated with the escape rate of the off-pathway oligomeric state.

Building Complexity: An In Vitro Study of Cytoplasmic Dynein with In Vivo Implications

Cytoplasmic dynein is the molecular motor responsible for most retrograde microtubule-based vesicular transport. In vitro single-molecule experiments suggest that dynein function is not as robust as that of kinesin-1 or myosin-V because dynein moves only a limited distance (approximately 800 nm) before detaching and can exert a modest (approximately 1 pN) force. However, dynein-driven cargos in vivo move robustly over many microns and exert forces of multiple pN. To determine how to go from limited single-molecule function to robust in vivo transport, we began to build complexity in a controlled manner by using in vitro experiments. We show that a single cytoplasmic dynein motor frequently transitions into an off-pathway unproductive state that impairs net transport. Addition of a second (and/or third) dynein motor, so that cargos are moved by two (or three) motors rather than one, is sufficient to recover several properties of in vivo motion; such properties include long cargo travels, robust motion, and increased forces. Part of this improvement appears to arise from selective suppression of the unproductive state of dynein rather than from a fundamental change in dynein's mechanochemical cycle. Multiple dyneins working together suppress shortcomings of a single motor and generate robust motion under in vitro conditions. There appears to be no need for additional cofactors (e.g., dynactin) for this improvement. Because cargos are often driven by multiple dyneins in vivo, our results show that changing the number of dynein motors could allow modulation of dynein function from the mediocre single-dynein limit to robust in vivo-like dynein-driven motion.

Antagonism, non-native interactions and non-two-state folding in S6 revealed by double-mutant cycle analysis

When folding to the native state N in the presence of salt, the apparent two-state folder S6 transiently forms a transient off-pathway state C with substantial secondary and tertiary structure. Fifteen double mutant cycles were analysed to compare side-chain interaction energies DeltaDeltaG(int) in C, N and TS (the transition state between N and the denatured state). The kinetic signatures of these destabilizing mutants suggest folding scenarios involving unfolding intermediates and even alternative unfolding pathways. However, restricting the kinetic data to linear parts of the chevron plot allows reliable extrapolation to zero molar denaturant of rate constants of folding, unfolding and misfolding. Side-chain interactions appear to contribute to the stability of C, but in a substantially non-native environment, as shown by changes in the sign of DeltaDeltaG(int) between C and N. Remarkably, there appear to be significant (0.7-2 kcal/mol) antagonistic interactions between the two residues Leu30 and Leu75 in N and TS, which may be linked to subtle structural changes seen in the crystal structures of the mutants. A small number of overlapping residues are involved in these kinds of antagonistic interactions in N, TS and C, suggesting that repulsive interactions are coded into the protein topology whether the protein folds or misfolds. Destabilizing double mutants indicate that apparent two-state folders can be induced to behave in more complex ways provided that the native state is suitably destabilized.

Kinetic Partitioning During Folding of the p53 DNA Binding Domain

The DNA-binding domain (DBD) of wild-type p53 loses DNA binding activity spontaneously at 37 degrees C in vitro, despite being thermodynamically stable at this temperature. We test the hypothesis that this property is due to kinetic misfolding of DBD. Interrupted folding experiments and chevron analysis show that native molecules are formed via four tracks (a-d) under strongly native conditions. Folding half-lives of tracks a-d are 7.8 seconds, 50 seconds, 5.3 minutes and more than five hours, respectively, in 0.3M urea (10 degrees C). Approximately equal fractions of molecules fold through each track in zero denaturant, but above 2.0M urea approximately 90% fold via track c. A kinetic mechanism consisting of two parallel folding channels (fast and slow) is proposed. Each channel populates an on-pathway intermediate that can misfold to form an aggregation-prone, dead-end species. Track a represents direct folding through the fast channel. Track b proceeds through the fast channel but via the off-pathway state. Track c corresponds to folding via the slow channel, primarily through the off-pathway state. Track d proceeds by way of an even slower, uncharacterized route. We postulate that activity loss is caused by partitioning to the slower tracks, and that structural unfolding limits this process. In support of this view, tumorigenic hot-spot mutants G245S, R249S and R282Q accelerate unfolding rates but have no affect on folding kinetics. We suggest that these and other destabilizing mutants facilitate loss of p53 function by causing DBD to cycle unusually rapidly between folded and unfolded states. A significant fraction of DBD molecules become effectively trapped in a non-functional state with each unfolding-folding cycle.