Urea modulation of beta-amyloid fibril growth: experimental studies and kinetic models

Aggregation of beta-amyloid (Abeta) into fibrillar deposits is widely believed to initiate a cascade of adverse biological responses associated with Alzheimer's disease. Although it was once assumed that the mature fibril was the toxic form of Abeta, recent evidence supports the hypothesis that Abeta oligomers, intermediates in the fibrillogenic pathway, are the dominant toxic species. In this work we used urea to reduce the driving force for Abeta aggregation, in an effort to isolate stable intermediate species. The effect of urea on secondary structure, size distribution, aggregation kinetics, and aggregate morphology was examined. With increasing urea concentration, beta-sheet content and the fraction of aggregated peptide decreased, the average size of aggregates was reduced, and the morphology of aggregates changed from linear to a globular/linear mixture and then to globular. The data were analyzed using a previously published model of Abeta aggregation kinetics. The model and data were consistent with the hypothesis that the globular aggregates were intermediates in the amyloidogenesis pathway rather than alternatively aggregated species. Increasing the urea concentration from 0.4 M to 2 M decreased the rate of filament initiation the most; between 2 M and 4 M urea the largest change was in partitioning between the nonamyloid and amyloid pathways, and between 4 M and 6 M urea, the most significant change was a reduction in the rate of filament elongation.     

Identification and characterization of key kinetic intermediates in amyloid beta-protein fibrillogenesis

Amyloid beta-protein (Abeta) assembly into toxic oligomeric and fibrillar structures is a seminal event in Alzheimer's disease, therefore blocking this process could have significant therapeutic benefit. A rigorous mechanistic understanding of Abeta assembly would facilitate the targeting and design of fibrillogenesis inhibitors. Prior studies have shown that Abeta fibrillogenesis involves conformational changes leading to the formation of extended beta-sheets and that an alpha-helix-containing intermediate may be involved. However, the significance of this intermediate has been a matter of debate. We report here that the formation of an oligomeric, alpha-helix-containing assembly is a key step in Abeta fibrillogenesis. The generality of this phenomenon was supported by conformational studies of 18 different Abeta peptides, including wild-type Abeta(1-40) and Abeta(1-42), biologically relevant truncated and chemically modified Abeta peptides, and Abeta peptides causing familial forms of cerebral amyloid angiopathy. Without exception, fibrillogenesis of these peptides involved an oligomeric alpha-helix-containing intermediate and the kinetics of formation of the intermediate and of fibrils was temporally correlated. The kinetics varied depending on amino acid sequence and the extent of peptide N- and C-terminal truncation. The pH dependence of helix formation suggested that Asp and His exerted significant control over this process and over fibrillogenesis in general. Consistent with this idea, Abeta peptides containing Asp-->Asn or His-->Gln substitutions showed altered fibrillogenesis kinetics. These data emphasize the importance of the dynamic interplay between Abeta monomer conformation and oligomerization state in controlling fibrillogenesis kinetics.     

Characterization of oligomers during alpha-synuclein aggregation using intrinsic tryptophan fluorescence

The aggregation of the presynaptic protein alpha-synuclein is associated with Parkinson's disease (PD). The details of the mechanism of aggregation, as well as the cytotoxic species, are currently not well understood. alpha-Synuclein has four tyrosine and no tryptophan residues. We introduced a tyrosine to tryptophan mutation at position 39 to create an intrinsic fluorescence probe and allow additional characterization of the aggregation process. Y39W alpha-synuclein had similar fibrillation kinetics (2-fold slower), pH-induced conformational changes, and fibril morphology to wild-type alpha-synuclein. In addition to intrinsic Trp fluorescence, acrylamide quenching, fluorescence anisotropy, ANS binding, dynamic light scattering, and FTIR were employed to monitor the kinetics of aggregation. These biophysical probes revealed the significant population of two classes of oligomeric intermediates, one formed during the lag period of fibrillation and the other present at the completion of fibrillation. As expected for a natively unfolded protein, Trp 39 was highly solvent-exposed in the monomer and is solvent-exposed in the two oligomeric intermediates; however, it is partially, but not fully, buried in the fibrils. These observations demonstrate the utility of Trp fluorescence labeled alpha-synuclein and demonstrate the existence of an oligomeric intermediate that exists as a transient reservoir of alpha-synuclein for fibrillation.     

On the Characterization of Intermediates in the Isodesmic Aggregation Pathway of Hen Lysozyme at Alkaline pH

Protein aggregation leading to formation of amyloid fibrils is a symptom of several diseases like Alzheimer’s, type 2 diabetes and so on. Elucidating the poorly understood mechanism of such phenomena entails the difficult task of characterizing the species involved at each of the multiple steps in the aggregation pathway. It was previously shown by us that spontaneous aggregation of hen-eggwhite lysozyme (HEWL) at room temperature in pH 12.2 is a good model to study aggregation. Here in this paper we investigate the growth kinetics, structure, function and dynamics of multiple intermediate species populating the aggregation pathway of HEWL at pH 12.2. The different intermediates were isolated by varying the HEWL monomer concentration in the 300 nM—0.12 mM range. The intermediates were characterized using techniques like steady-state and nanosecond time-resolved fluorescence, atomic force microscopy and dynamic light scattering. Growth kinetics of non-fibrillar HEWL aggregates were fitted to the von Bertalanffy equation to yield a HEWL concentration independent rate constant (k = (6.6±0.6)×10⁻⁵ s⁻¹). Our results reveal stepwise changes in size, molecular packing and enzymatic activity among growing HEWL aggregates consistent with an isodesmic aggregation model. Formation of disulphide bonds that crosslink the monomers in the aggregate appear as a unique feature of this aggregation. AFM images of multiple amyloid fibrils emanating radially from amorphous aggregates directly confirmed that on-pathway fibril formation was feasible under isodesmic polymerization. The isolated HEWL aggregates are revealed as polycationic protein nanoparticles that are robust at neutral pH with ability to take up non-polar molecules like ANS.     

Kinetic analysis of beta-amyloid fibril elongation

We used surface plasmon resonance biosensors to evaluate the kinetics associated with the initial events of beta-amyloid (Abeta) fibril elongation. Fibrils were immobilized on the sensor chip surface and extended by exposure to soluble Abeta(1-40) peptide. The fibril surfaces bound Congo red, a marker for beta sheet structures, and exhibited a slow linear background decay that is consistent with fibril depolymerization. Sonicated fibrils supported elongation better than unsonicated fibrils, which is consistent with fibril extension reactions. The kinetic data revealed that peptide association and dissociation occurred in multiple steps. Kinetic rate constants for fibril extension were determined by globally fitting the response data with a three-step polymerization model. In the first step, the soluble peptide binds to the growing fibril tip in a readily reversible reaction. The subsequent steps likely allow bound peptide to be stabilized into the growing fiber through postbinding transitional events. Using a mutant peptide, F19P Abeta(1-40), we illustrate how the biosensor assay can be used to probe structure/function relationships of fibril elongation.     

Protofibril formation of amyloid beta-protein at low pH via a non-cooperative elongation mechanism

Deposition of the amyloid beta-protein (Abeta) in senile or diffuse plaques is a distinctive feature of Alzheimer's disease. The role of Abeta aggregates in the etiology of the disease is still controversial. The formation of linear aggregates, known as amyloid fibrils, has been proposed as the onset and the cause of pathological deposition. Yet, recent findings suggest that a more crucial role is played by prefibrillar oligomeric assemblies of Abeta that are highly toxic in the extracellular environment. In the present work, the mechanism of protofibril formation is studied at pH 3.1, starting from a solution of oligomeric precursors. By combining static light scattering and photon correlation spectroscopy, the growth of the mass and the size of aggregates are determined at different temperatures. Analysis and scaling of kinetic data reveal that under the studied conditions protofibrils are formed via a single non-cooperative elongation mechanism, not prompted by nucleation. This process is well described as a linear colloidal aggregation due to diffusion and coalescence of growing aggregates. The rate of elongation follows an Arrhenius law with an activation enthalpy of 15 kcal mol(-1). Such a value points to a conformational change of peptides or oligomers being involved in binding to protofibrils or in general to a local reorganization of each aggregate. These results contribute to establishing a clearer relation at the molecular level between the fibrillation mechanism and fibrillar precursors. The observation of a non-cooperative aggregation pathway supports the hypothesis that amyloid formation may represent an escape route from a dangerous condition, induced by the presence of toxic oligomeric species.     

Elucidation of the molecular mechanism during the early events in immunoglobulin light chain amyloid fibrillation. Evidence for an off-pathway oligomer at acidic pH

Light chain amyloidosis involves the systemic pathologic deposition of monoclonal light chain variable domains of immunoglobulins as insoluble fibrils. The variable domain LEN was obtained from a patient who had no overt amyloidosis; however, LEN forms fibrils in vitro, under mildly destabilizing conditions. The in vitro kinetics of fibrillation were investigated using a wide variety of probes. The rate of fibril formation was highly dependent on the initial protein concentration. In contrast to most amyloid systems, the kinetics became slower with increasing LEN concentrations. At high protein concentrations a significant lag in time was observed between the conformational changes and the formation of fibrils, consistent with the formation of soluble off-pathway oligomeric species and a branched pathway. The presence of off-pathway species was confirmed by small angle x-ray scattering. At low protein concentrations the structural rearrangements were concurrent with fibril formation, indicating the absence of formation of the off-pathway species. The data are consistent with a model for fibrillation in which a dimeric form of LEN (at high protein concentration) inhibits fibril formation by interaction with an intermediate on the fibrillation pathway and leads to formation of the off-pathway intermediate.     

Kinetics of inhibition of beta-amyloid aggregation by transthyretin

Deposition of beta-amyloid (Abeta) fibrils is an early event in the neurodegenerative processes associated with Alzheimer's disease. According to the "amyloid cascade" hypothesis, Abeta aggregation, and its subsequent deposition as fibrils, is the underlying cause of disease. Abeta is a proteolytic product of amyloid precursor protein (APP); several mutations in APP have been identified that are associated with early onset of disease. Transgenic mice overexpressing APP with the Swedish mutation develop numerous plaques but, surprisingly, lack the neurofibrillary tangles and neuronal loss characteristic of Alzheimer's disease, in apparent contradiction of the amyloid cascade hypothesis. However, recent studies suggest that coproduction of sAPPalpha, an alternative proteolytic product of APP, increases synthesis of transthyretin that, in turn, interacts directly with Abeta to inhibit its toxicity. Here we report results from biophysical analysis of Abeta aggregation kinetics in the presence of transthryetin. At substoichiometric ratios, transthyretin drastically decreased the rate of aggregation without affecting the fraction of Abeta in the aggregate pool. Detailed analysis of the data using a mathematical model demonstrated that the decrease in aggregation rate was due to both a decrease in the rate of elongation relative to the rate of initiation of filaments and a decrease in lateral association of filaments to fibrils. Tryptophan quenching data indicated that transthyretin binds weakly to Abeta, with an estimated apparent KS of 2300 M-1. Taken together, the data support a hypothesis wherein transthyretin preferentially binds to aggregated rather than monomeric Abeta and arrests further growth of the aggregates.     

Distinct early folding and aggregation properties of Alzheimer amyloid-beta peptides Abeta40 and Abeta42: stable trimer or tetramer formation by Abeta42

The amyloid beta peptide (Abeta), composed of 40 or 42 amino acids, is a critical component in the etiology of the neurodegenerative Alzheimer disease. Abeta is prone to aggregate and forms amyloid fibrils progressively both in vitro and in vivo. To understand the process of amyloidogenesis, it is pivotal to examine the initial stages of the folding process. We examined the equilibrium folding properties, assembly states, and stabilities of the early folding stages of Abeta40 and Abeta42 prior to fibril formation. We found that Abeta40 and Abeta42 have different conformations and assembly states upon refolding from their unfolded ensembles. Abeta40 is predominantly an unstable and collapsed monomeric species, whereas Abeta42 populates a stable structured trimeric or tetrameric species at concentrations above approximately 12.5 microm. Thermodynamic analysis showed that the free energies of Abeta40 monomer and Abeta42 trimer/tetramer are approximately 1.1 and approximately 15/ approximately 22 kcal/mol, respectively. The early aggregation stages of Abeta40 and Abeta42 contain different solvent-exposed hydrophobic surfaces that are located at the sequences flanking its protease-resistant segment. The amyloidogenic folded structure of Abeta is important for the formation of spherical beta oligomeric species. However, beta oligomers are not an obligatory intermediate in the process of fibril formation because oligomerization is inhibited at concentrations of urea that have no effect on fibril formation. The distinct initial folding properties of Abeta40 and Abeta42 may play an important role in the higher aggregation potential and pathological significance of Abeta42.     

Conversion of non-fibrillar beta-sheet oligomers into amyloid fibrils in Alzheimer's disease amyloid peptide aggregation

Abeta(1-40) is one of the main components of the fibrils found in amyloid plaques, a hallmark of brains affected by Alzheimer's disease. It is known that prior to the formation of amyloid fibrils in which the peptide adopts a well-ordered intermolecular beta-sheet structure, peptide monomers associate forming low and high molecular weight oligomers. These oligomers have been previously described in electron microscopy, AFM, and exclusion chromatography studies. Their specific secondary structures however, have not yet been well established. A major problem when comparing aggregation and secondary structure determinations in concentration-dependent processes such as amyloid aggregation is the different concentration range required in each type of experiment. In the present study we used the dye Thioflavin T (ThT), Fourier-transform infrared spectroscopy, and electron microscopy in order to structurally characterize the different aggregated species which form during the Abeta(1-40) fibril formation process. A unique sample containing 90microM peptide was used. The results show that oligomeric species which form during the lag phase of the aggregation kinetics are a mixture of unordered, helical, and intermolecular non-fibrillar beta-structures. The number of oligomers and the amount of non-fibrillar beta-structures grows throughout the lag phase and during the elongation phase these non-fibrillar beta-structures are transformed into fibrillar (amyloid) beta-structures, formed by association of high molecular weight intermediates.     

Kinetic studies of amyloid beta-protein fibril assembly. Differential effects of alpha-helix stabilization

Amyloid beta-protein (Abeta) fibril assembly is a defining characteristic of Alzheimer's disease. Fibril formation is a complex nucleation-dependent polymerization process characterized in vitro by an initial lag phase. To a significant degree, this phase is a consequence of the energy barrier that must be overcome in order for Abeta monomers to fold and oligomerize into fibril nuclei. Here we show that low concentrations of 2,2,2-trifluoroethanol (TFE) convert predominately unstructured Abeta monomers into partially ordered, quasistable conformers. Surprisingly, this results in a temporal decrease in the lag phase for fibril formation and a significant increase in the rate of fibril elongation. The TFE effect is concentration dependent and is maximal at approximately 20% (v/v). In the presence of low concentrations of TFE, fibril formation is observed in Abeta samples at nanomolar concentration, well below the critical concentration for Abeta fibril formation in the absence of TFE. As the amount of TFE is increased above 20%, helix content progressively rises to approximately 80%, a change paralleled first by a decrease in elongation rate and then by a complete cessation of fibril growth. These findings are consistent with the hypothesis that a partially folded helix-containing conformer is an intermediate in Abeta fibril assembly. The requirement that Abeta partially folds in order to assemble into fibrils contrasts with the mechanism of amyloidogenesis of natively folded proteins such as transthyretin and lysozyme, in which partial unfolding is a prerequisite. Our results suggest that in vivo, factors that affect helix formation and stability will have significant effects on the kinetics of Abeta fibril formation.     

Alternate aggregation pathways of the Alzheimer beta-amyloid peptide: Abeta association kinetics at endosomal pH

The deposition of beta-amyloid peptide (Abeta) fibrils around neurons is an invariable feature of Alzheimer's disease and there is increasing evidence that fibrillar deposits and/or prefibrillar intermediates play a central role in the observed neurodegeneration. One site of Abeta generation is the endosomes, and we have investigated the kinetics of Abeta association at endosomal pH over physiologically relevant time frames. We have identified three distinct Abeta association phases that occur at rates comparable to endosomal transit times. Rapid formation of burst phase aggregates, larger than 200nm, was observed within 15 seconds. Two slower association phases were detected by fluorescence resonance energy transfer and termed phase 1 and phase 2 aggregation reactions. At 20 microM Abeta, pH 6, the half lives of the phase 1 and phase 2 aggregation phases were 3.15 minutes and 17.66 minutes, respectively. Atomic force microscopy and dynamic light scattering studies indicate that the burst phase aggregate is large and amorphous, while phase 1 and 2 aggregates are spherical with hydrodynamic radii around 30 nm. There is an apparent equilibrium, potentially mediated through a soluble Abeta intermediate, between the large burst phase aggregates and phase 1 and 2 spherical particles. The large burst phase aggregates form quickly, however, they disappear as the equilibrium shifts toward the spherical aggregates. These aggregated species do not contain alpha-helical or beta-structure as determined by circular dichroism spectroscopy. However, after two weeks beta-structure is observed and is attributable to the insoluble portion of the sample. After two months, mature amyloid fibrils appear and the spherical aggregates are significantly diminished.     

Partially folded intermediates as critical precursors of light chain amyloid fibrils and amorphous aggregates

Light chain, or AL, amyloidosis is a pathological condition arising from systemic extracellular deposition of monoclonal immunoglobulin light chain variable domains in the form of insoluble amyloid fibrils, especially in the kidneys. Substantial evidence suggests that amyloid fibril formation from native proteins occurs via a conformational change leading to a partially folded intermediate conformation, whose subsequent association is a key step in fibrillation. In the present investigation, we have examined the properties of a recombinant amyloidogenic light chain variable domain, SMA, to determine whether partially folded intermediates can be detected and correlated with aggregation. The results from spectroscopic and hydrodynamic measurements, including far- and near-UV circular dichroism, FTIR, NMR, and intrinsic tryptophan fluorescence and small-angle X-ray scattering, reveal the build-up of two partially folded intermediate conformational states as the pH is decreased (low pH destabilized the protein and accelerated the kinetics of aggregation). A relatively nativelike intermediate, I(N), was observed between pH 4 and 6, with little loss of secondary structure, but with significant tertiary structure changes and enhanced ANS binding, indicating exposed hydrophobic surfaces. At pH below 3, we observed a relatively unfolded, but compact, intermediate, I(U), which was characterized by decreased tertiary and secondary structure. The I(U) intermediate readily forms amyloid fibrils, whereas I(N) preferentially leads to amorphous aggregates. Except at pH 2, where negligible amorphous aggregate is formed, the amorphous aggregates formed significantly more rapidly than the fibrils. This is the first indication that different partially folded intermediates may be responsible for different aggregation pathways (amorphous and fibrillar). The data support the hypothesis that amyloid fibril formation involves the ordered self-assembly of partially folded species that are critical soluble precursors of fibrils.     

Mechanism of accelerated assembly of beta-amyloid filaments into fibrils by KLVFFK(6)

Extracellular senile plaques are a central pathological feature of Alzheimer's disease. At the core of these plaques are fibrillar deposits of beta-amyloid peptide (Abeta). In vitro, Abeta spontaneously assembles into amyloid fibrils of cross-beta sheet structure. Although it was once believed that the fibrils themselves were toxic, more recent data supports the hypothesis that aggregation intermediates, rather than fully formed fibrils, are the most damaging to neuronal tissue. In previously published work, we identified several small peptides that interact with Abeta and increase its aggregation rate while decreasing its toxicity. In this work, we examined in detail the interaction between Abeta and one of these peptides. Using a mathematical model of Abeta aggregation kinetics, we show that the dominant effect of the peptide is to accelerate lateral association of Abeta filaments into fibrils.     

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.     

Probing the mechanism of insulin fibril formation with insulin mutants.

The molecular basis of insulin fibril formation was investigated by studying the structural properties and kinetics of fibril formation of 20 different human insulin mutants at both low pH (conditions favoring monomer/dimer) and at pH 7.4 (conditions favoring tetramer/hexamer). Small-angle X-ray scattering showed insulin to be monomeric in 20% acetic acid, 0.1 M NaCl, pH 2. The secondary structure of the mutants was assessed using far-UV circular dichroism, and the tertiary structure was determined using near-UV circular dichroism, quenching of intrinsic fluorescence by acrylamide and interactions with the hydrophobic probe 1-anilino-8-naphthalene-sulfonic acid (ANS). The kinetics of fibril formation were monitored with the fluorescent dye, Thioflavin T. The results indicate that the monomer is the state from which fibrils arise, thus under some conditions dissociation of hexamers may be rate limiting or partially rate limiting. The insulin mutants were found to retain substantial nativelike secondary and tertiary structure under all conditions studied. The results suggest that fibril formation of the insulin mutants is controlled by specific molecular interactions that are sensitive to variations in the primary structure. The observed effects of several mutations on the rate of fibril formation are inconsistent with a previously suggested model for fibrillation [Brange, J., Whittingham, J., Edwards, D., Youshang, Z., Wollmer, A., Brandenburg, D., Dodson, G., and Finch, J. (1997) Curr. Sci. 72, 470-476]. Two surfaces on the insulin monomer are identified as potential interacting sites in insulin fibrils, one consisting of the residues B10, B16, and B17 and the other consisting of at least the residues A8 and B25. The marked increase in the lag time for fibril formation with mutations to more polar residues, as well as mutations to charged residues, demonstrates the importance of both hydrophobic and electrostatic interactions in the initial stages of fibrillation. A model for insulin fibril formation is proposed in which the formation of a partially folded intermediate is the precursor for associated species on the pathway to fibril formation.     

The kinetics of aggregation of poly-glutamic acid based polypeptides

The aggregation of two negatively-charged polypeptides, poly-L-glutamic acid (PE) and a copolymer of poly-glutamic acid and poly-alanine (PEA), has been studied at different peptide and salt concentrations and solution pH conditions. The kinetics of aggregation were based on Thioflavin T (ThT) fluorescence measurements. The observed lag phase shortened and the aggregation was faster as the pH approached the polypeptides' isoelectric points. While the initial polypeptide structures of PE and PEA appeared identical as determined from circular dichroism spectroscopy, the final aggregate morphology differed; PE assumed large twisted lamellar structures and the PEA formed typical amyloid-like fibrils, although both contained extensive beta-sheet structure. Differences in aggregation behavior were observed for the two polypeptides as a function of salt concentration; aggregation progressed more slowly for PE and more quickly for PEA with increasing salt concentration. Several models of aggregation kinetics were fit to the data. No model yielded consistent rate constants or a critical nucleus size. A modified nucleated polymerization model was developed based on that of Powers and Powers [E.T. Powers, D.L. Powers, The kinetics of nucleated polymerizations at high concentrations: Amyloid fibril formation near and above the "supercritical concentration", Biophys. J. 91 (2006) 122-132], which incorporated the ability of oligomeric species to interact. This provided a best fit to the experimental data.     

Transthyretin aggregation under partially denaturing conditions is a downhill polymerization

The deposition of fibrils and amorphous aggregates of transthyretin (TTR) in patient tissues is a hallmark of TTR amyloid disease, but the molecular details of amyloidogenesis are poorly understood. Tetramer dissociation is typically rate-limiting for TTR amyloid fibril formation, so we have used a monomeric variant of TTR (M-TTR) to study the mechanism of aggregation. Amyloid formation is often considered to be a nucleation-dependent process, where fibril growth requires the formation of an oligomeric nucleus that is the highest energy species on the pathway. According to this model, the rate of fibril formation should be accelerated by the addition of preformed aggregates or "seeds", which effectively bypasses the nucleation step. Herein, we demonstrate that M-TTR amyloidogenesis at low pH is a complex, multistep reaction whose kinetic behavior is incompatible with the expectations for a nucleation-dependent polymerization. M-TTR aggregation is not accelerated by seeding, and the dependence of the reaction timecourse is first-order on the M-TTR concentration, consistent either with a dimeric nucleus or with a nonnucleated process where each step is bimolecular and essentially irreversible. These studies suggest that amyloid formation by M-TTR under partially denaturing conditions is a downhill polymerization, in which the highest energy species is the native monomer. Our results emphasize the importance of therapeutic strategies that stabilize the TTR tetramer and may help to explain why more than eighty TTR variants are disease-associated. The differences between amyloid formation by M-TTR and other amyloidogenic peptides (such as amyloid beta-peptide and islet amyloid polypeptide) demonstrate that these polypeptides do not share a common aggregation mechanism, at least under the conditions examined thus far.