Heat-induced conversion of beta(2)-microglobulin and hen egg-white lysozyme into amyloid fibrils

Thermodynamic parameters characterizing protein stability can be obtained for a fully reversible folding/unfolding system directly by differential scanning calorimetry (DSC). However, the reversible DSC profile can be altered by an irreversible step causing aggregation. Here, to obtain insight into amyloid fibrils, ordered and fibrillar aggregates responsible for various amyloidoses, we studied the effects on human beta(2)-microglobulin and hen egg-white lysozyme of a combination of agitation and heating. Aggregates formed by mildly agitating protein solutions in the native state in the presence of NaCl were heated in the cell of the DSC instrument. For beta(2)-microglobulin, with an increase in the concentration of NaCl at neutral pH, the thermogram began to show an exothermic transition accompanied by a large decrease in heat capacity, followed by a kinetically controlled thermal response. Similarly, the aggregated lysozyme at a high concentration of NaCl revealed a similar distinct transition in the DSC thermogram over a wide pH range. Electron microscopy demonstrated the conformational change into amyloid fibrils. Taken together, the combined use of agitation and heating is a powerful way to generate amyloid fibrils from two proteins, beta(2)-microglobulin and hen egg-white lysozyme, and to evaluate the effects of heat on fibrillation, in which the heat capacity is crucial to characterizing the transition.     

Structural studies reveal that the diverse morphology of β2-microglobulin aggregates is a reflection of different molecular architectures

Amyloid deposition accompanies over 20 degenerative diseases in human, including Alzheimer's, Parkinson's, and prion diseases. Recent studies revealed the importance of other type of protein aggregates, e.g., non-specific aggregates, protofibrils, and small oligomers in the development of such diseases and proved their increased toxicity for living cells in comparison with mature amyloid fibrils. We carried out a comparative structural analysis of different monomeric and aggregated states of β₂-microglobulin, a protein responsible for hemodialysis-related amyloidosis. We investigated the structure of the native and acid-denatured states, as well as that of mature fibrils, immature fibrils, amorphous aggregates, and heat-induced filaments, prepared under various in vitro conditions. Infrared spectroscopy demonstrated that the β-sheet compositions of immature fibrils, heat-induced filaments and amorphous aggregates are characteristic of antiparallel intermolecular β-sheet structure while mature fibrils are different from all others suggesting a unique overall structure and assembly. Filamentous aggregates prepared by heat treatment are of importance in understanding the in vivo disease because of their stability under physiological conditions, where amyloid fibrils and protofibrils formed at acidic pH depolymerize. Atomic force microscopy of heat-induced filaments represented a morphology similar to that of the low pH immature fibrils. At a pH close to the pI of the protein, amorphous aggregates were formed readily with association of the molecules in native-like conformation, followed by formation of intermolecular β-sheet structure in a longer time-scale. Extent of the core buried from the solvent in the various states was investigated by H/D exchange of the amide protons.     

Conformational stability of amyloid fibrils of beta2-microglobulin probed by guanidine-hydrochloride-induced unfolding

Although the stability of globular proteins has been studied extensively, that of amyloid fibrils is scarcely characterized. Beta2-microglobulin (beta2-m) is a major component of the amyloid fibrils observed in patients with dialysis-related amyloidosis. We studied the effects of guanidine hydrochloride on the amyloid fibrils of beta2-m, revealing a cooperative unfolding transition similar to that of the native state. The stability of amyloid fibrils increased on the addition of ammonium sulfate, consistent with a role of hydrophobic interactions. The results indicate that the analysis of unfolding transition is useful to obtain insight into the structural stability of amyloid fibrils.     

Structural stability of amyloid fibrils of beta(2)-microglobulin in comparison with its native fold

Among various amyloidogenic proteins, beta(2)-microglobulin (beta2-m) responsible for dialysis-related amyloidosis is a target of extensive study because of its clinical importance and suitable size for examining the formation of amyloid fibrils in comparison with protein folding to the native state. The structure and stability of amyloid fibrils have been studied with various physicochemical methods, including H/D exchange of amyloid fibrils combined with dissolution of fibrils by dimethylsulfoxide and NMR analysis, thermodynamic analysis of amyloid fibril formation by isothermal calorimetry, and analysis of the effects of pressure on the structure of amyloid fibrils. The results are consistent with the view that amyloid fibrils are a main-chain-dominated structure with larger numbers of hydrogen bonds and pressure-accessible cavities in the interior, in contrast to the side-chain-dominated native structure with the optimal packing of amino acid residues. We consider that a main-chain dominated structure provides the structural basis for various conformational states even with one protein. When this feature is combined with another unique feature, template-dependent growth, propagation and maturation of the amyloid conformation, which cannot be predicted with Anfinsen's dogma, take place.     

Exothermic effects observed upon heating of beta2-microglobulin monomers in the presence of amyloid seeds

To understand the initial stages in the formation of amyloid fibrils of beta(2)-microglobulin, a protein responsible for dialysis-related amyloidosis, the effects of heat on the acid-unfolded monomer at pH 2.5 were studied. In the presence of a low concentration of seed fibrils, differential scanning calorimetric thermograms of acid-unfolded beta(2)-microglobulin monomers showed a large decrease in heat capacity with a sigmoidal temperature-dependence, which was subsequently released at higher temperature. Measurements of circular dichroism, atomic force microscopy, ultracentrifugation, and repeated differential scanning calorimetry indicated that the exothermic sigmoidal transition is accompanied by the conversion of about 12% of the monomeric beta(2)-microglobulin molecules into amyloid fibrils, which subsequently dissociate into monomers at high temperature. Interestingly, amyloid fibrils, formed partly after the sigmoidal transition, exhibited a heating rate-dependent, kinetically controlled thermal response, indicating that 12% of the total protein is enough to exhibit the unique thermal response. On the other hand, the salt-induced protofibrils did not show such a calorimetric response, indicating that the kinetic thermal response is unique to the particular structure of fibrils. Taken together, although the calorimetric behavior of amyloid fibrils remains elusive, it may be interpreted in terms of the effects of heat associated with the formation, the association, and the unfolding of fibrils, in which the interactions between specific beta-sheet structures and water molecules play a crucial role and are sensitively reflected in the heat capacity change in protein solution.     

Structural stability of amyloid fibrils of β2-microglobulin in comparison with its native fold

Among various amyloidogenic proteins, β₂-microglobulin (β2-m) responsible for dialysis-related amyloidosis is a target of extensive study because of its clinical importance and suitable size for examining the formation of amyloid fibrils in comparison with protein folding to the native state. The structure and stability of amyloid fibrils have been studied with various physicochemical methods, including H/D exchange of amyloid fibrils combined with dissolution of fibrils by dimethylsulfoxide and NMR analysis, thermodynamic analysis of amyloid fibril formation by isothermal calorimetry, and analysis of the effects of pressure on the structure of amyloid fibrils. The results are consistent with the view that amyloid fibrils are a main-chain-dominated structure with larger numbers of hydrogen bonds and pressure-accessible cavities in the interior, in contrast to the side-chain-dominated native structure with the optimal packing of amino acid residues. We consider that a main-chain dominated structure provides the structural basis for various conformational states even with one protein. When this feature is combined with another unique feature, template-dependent growth, propagation and maturation of the amyloid conformation, which cannot be predicted with Anfinsen's dogma, take place.     

Kinetically controlled thermal response of beta2-microglobulin amyloid fibrils

Calorimetric measurements were carried out using a differential scanning calorimeter in the temperature range from 10 to 120 degrees C for characterizing the thermal response of beta2-microglobulin amyloid fibrils. The thermograms of amyloid fibril solution showed a remarkably large decrease in heat capacity that was essentially released upon the thermal unfolding of the fibrils, in which the magnitude of negative heat capacity change was not explicable in terms of the current accessible surface area model of protein structural thermodynamics. The heat capacity-temperature curve of amyloid fibrils prior to the fibril unfolding exhibited an unusual dependence on the fibril concentration and the heating rate. Particularly, the heat needed to induce the thermal response was found to be linearly dependent on the heating rate, indicating that its thermal response is under a kinetic control and precluding the interpretation in terms of equilibrium thermodynamics. Furthermore, amyloid fibrils of amyloid beta peptides also exhibited a heating rate-dependent exothermic process before the fibril unfolding, indicating that the kinetically controlled thermal response may be a common phenomenon to amyloid fibrils. We suggest that the heating rate-dependent negative change in heat capacity is coupled to the association of amyloid fibrils with characteristic hydration pattern.     

A systematic investigation into the effect of protein destabilisation on beta 2-microglobulin amyloid formation

Beta-2-microglobulin (beta(2)m) has been shown to form amyloid fibrils with distinct morphologies under acidic conditions in vitro. Short, curved fibrils (<600 nm in length), form rapidly without a lag phase, with a maximum rate at pH 3.5. By contrast, fibrils with a long (approximately 1 microm), straight morphology are produced by incubation of the protein at pH< or =3.0. Both fibril types display Congo red birefringence, bind Thioflavin-T and have X-ray fibre diffraction patterns consistent with a cross-beta structure. In order to investigate the role of different partially folded states in generating fibrils of each type, and to probe the effect of protein stability on amyloid formation, we have undertaken a detailed mutagenesis study of beta(2)m. Thirteen variants containing point mutations in different regions of the native protein were created and their structure, stability and fibril forming propensities were investigated as a function of pH. By altering the stability of the native protein in this manner, we show that whilst destabilisation of the native state is important in the generation of amyloid fibrils, population of specific denatured states is a pre-requisite for amyloid formation from this protein. Moreover, we demonstrate that the formation of fibrils with different morphologies in vitro correlates with the relative population of different precursor states.     

Optimum amyloid fibril formation of a peptide fragment suggests the amyloidogenic preference of beta2-microglobulin under physiological conditions

Beta(2)-microglobulin (beta(2)m) is a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. Although full-length beta(2)m readily forms amyloid fibrils in vitro by seed-dependent extension with a maximum at pH 2.5, fibril formation under physiological conditions as detected in patients has been difficult to reproduce. A 22-residue K3 peptide of beta(2)m, Ser(20)-Lys(41), obtained by digestion with Acromobacter protease I, forms amyloid fibrils without seeding. To obtain further insight into the mechanism of fibril formation, we studied the pH dependence of fibril formation of the K3 peptide and its morphology using a ThT fluorescence assay and electron microscopy, respectively. K3 peptide formed amyloid fibrils over a wide range of pH values with an optimum around pH 7 and contrasted with the pH profile of the seed-dependent extension reaction of full-length beta(2)m. This suggests that once the rigid native-fold of beta(2)m is unfolded and additional factors triggering the nucleation process are provided, full-length beta(2)m discloses an intrinsic potential to form amyloid fibrils at neutral pH. The fibril formation was strongly promoted by dimerization of K3 through Cys(25). The morphology of the fibrils varied depending on the fibril formation conditions and the presence or absence of a disulfide bond. Various fibrils had the potential to seed fibril formation of full-length beta(2)m accompanied with a characteristic lag phase, suggesting that the internal structures are similar.     

Structural studies reveal that the diverse morphology of beta(2)-microglobulin aggregates is a reflection of different molecular architectures

Amyloid deposition accompanies over 20 degenerative diseases in human, including Alzheimer's, Parkinson's, and prion diseases. Recent studies revealed the importance of other type of protein aggregates, e.g., non-specific aggregates, protofibrils, and small oligomers in the development of such diseases and proved their increased toxicity for living cells in comparison with mature amyloid fibrils. We carried out a comparative structural analysis of different monomeric and aggregated states of beta(2)-microglobulin, a protein responsible for hemodialysis-related amyloidosis. We investigated the structure of the native and acid-denatured states, as well as that of mature fibrils, immature fibrils, amorphous aggregates, and heat-induced filaments, prepared under various in vitro conditions. Infrared spectroscopy demonstrated that the beta-sheet compositions of immature fibrils, heat-induced filaments and amorphous aggregates are characteristic of antiparallel intermolecular beta-sheet structure while mature fibrils are different from all others suggesting a unique overall structure and assembly. Filamentous aggregates prepared by heat treatment are of importance in understanding the in vivo disease because of their stability under physiological conditions, where amyloid fibrils and protofibrils formed at acidic pH depolymerize. Atomic force microscopy of heat-induced filaments represented a morphology similar to that of the low pH immature fibrils. At a pH close to the pI of the protein, amorphous aggregates were formed readily with association of the molecules in native-like conformation, followed by formation of intermolecular beta-sheet structure in a longer time-scale. Extent of the core buried from the solvent in the various states was investigated by H/D exchange of the amide protons.     

Amyloid-like fibrils from a domain-swapping protein feature a parallel, in-register conformation without native-like interactions

The formation of amyloid-like fibrils is characteristic of various diseases, but the underlying mechanism and the factors that determine whether, when, and how proteins form amyloid, remain uncertain. Certain mechanisms have been proposed based on the three-dimensional or runaway domain swapping, inspired by the fact that some proteins show an apparent correlation between the ability to form domain-swapped dimers and a tendency to form fibrillar aggregates. Intramolecular β-sheet contacts present in the monomeric state could constitute intermolecular β-sheets in the dimeric and fibrillar states. One example is an amyloid-forming mutant of the immunoglobulin binding domain B1 of streptococcal protein G, which in its native conformation consists of a four-stranded β-sheet and one α-helix. Under native conditions this mutant adopts a domain-swapped dimer, and it also forms amyloid-like fibrils, seemingly in correlation to its domain-swapping ability. We employ magic angle spinning solid-state NMR and other methods to examine key structural features of these fibrils. Our results reveal a highly rigid fibril structure that lacks mobile domains and indicate a parallel in-register β-sheet structure and a general loss of native conformation within the mature fibrils. This observation contrasts with predictions that native structure, and in particular intermolecular β-strand interactions seen in the dimeric state, may be preserved in "domain-swapping" fibrils. We discuss these observations in light of recent work on related amyloid-forming proteins that have been argued to follow similar mechanisms and how this may have implications for the role of domain-swapping propensities for amyloid formation.     

Flow-induced alignment of amyloid protofilaments revealed by linear dichroism

Amyloid fibrils underlying various serious amyloidoses including Alzheimer and prion diseases form characteristic deposits in which linear fibrils with an unbranched and rigid morphology associate laterally or radially, e.g. radial senile amyloid plaques of amyloid beta. To clarify the formation of these high order amyloid deposits, studying the rheology is important. A 22-residue K3 peptide fragment of beta2-microglobulin, a protein responsible for dialysis-related amyloidosis, forms long and homogeneous protofilament-like fibrils in 20% (v/v) 2,2,2-trifluoroethanol and 10 mM HCl (pH approximately 2). Here, using circular dichroism and linear dichroism, we observed the flow-induced alignment of fibrils. Analysis of far- and near-UV linear dichroism spectra suggested that both the net pi-pi* transition moment of the backbone carbonyl group and L(b) transition moment of the Tyr(26) side chain are oriented in parallel to the fibril axis, revealing the structural details of amyloid protofilaments. Moreover, the intensities of flow-induced circular dichroism or linear dichroism signals depended critically on the length and type of fibrils, suggesting that they are useful for detecting and characterizing amyloid fibrils.     

Amyloid nucleation triggered by agitation of beta2-microglobulin under acidic and neutral pH conditions

Amyloid nucleation through agitation was studied with beta2-microglobulin, which is responsible for dialysis-related amyloidosis, in the presence of salt under acid and neutral pH conditions. First, the aggregation of beta2-microglobulin in NaCl solutions was achieved by mildly agitating for 24 h at 37 degrees C protein solutions in three different states: acid-unfolded, salt-induced protofibrillar, and native. The formation of aggregates was confirmed by an increase in light scattering intensity of the solutions. Then, the aggregated samples were incubated without agitation at 37 degrees C for up to 25-45 days. The structural changes in the aggregated state during the incubation period were examined by means of fluorescence spectroscopy with thioflavin T, circular dichroism spectroscopy, and electron microscopy. The results revealed that all the samples in the different states produced a mature amyloid nucleus upon agitation, after which the fibrils elongated without any detectable lag phase during the incubation, with the acid-unfolded protein better suited to undergoing the structural rearrangements necessary to form amyloid fibrils than the more structured forms. The amount of aggregate including the amyloid nucleus produced by agitation from the native conformation at neutral pH was estimated to be about 9% of all the protein by an analysis using ultracentrifugation. Additionally, amyloid nucleation by agitation was similarly achieved for a different protein, hen egg-white lysozyme, in 0.5 M NaCl solution at neutral pH. Taken together, the agitation-treated aggregates of both proteins have a high propensity to produce an amyloid nucleus even at neutral pH, providing evidence that the aggregation pathway involves amyloid nucleation under entirely native conditions.     

Oligomeric States along the Folding Pathways of β2-Microglobulin: Kinetics,Thermodynamics, and Structure

The transition of proteins from their soluble functional state to amyloid fibrils and aggregates is associated with the onset of several human diseases. Protein aggregation often requires some structural reshaping and the subsequent formation of intermolecular contacts. Therefore, the study of the conformation of excited protein states and their ability to form oligomers is of primary importance for understanding the molecular basis of amyloid fibril formation. Here, we investigated the oligomerization processes that occur along the folding of the amyloidogenic human protein β2-microglobulin. The combination of real-time two-dimensional NMR data with real-time small-angle X-ray scattering measurements allowed us to derive thermodynamic and kinetic information on protein oligomerization of different conformational states populated along the folding pathways. In particular, we could demonstrate that a long-lived folding intermediate (I-state) has a higher propensity to oligomerize compared to the native state. Our data agree well with a simple five-state kinetic model that involves only monomeric and dimeric species. The dimers have an elongated shape with the dimerization interface located at the apical side of β2-microglobulin close to Pro32, the residue that has a trans conformation in the I-state and a cis conformation in the native (N) state. Our experimental data suggest that partial unfolding in the apical half of the protein close to Pro32 leads to an excited state conformation with enhanced propensity for oligomerization. This excited state becomes more populated in the transient I-state due to the destabilization of the native conformation by the trans-Pro32 configuration.     

Mapping the core of the beta(2)-microglobulin amyloid fibril by H/D exchange

Despite numerous efforts, the lack of detailed structural information on amyloid fibrils has hindered clarification of the mechanism of their formation. Here, we describe a novel procedure for characterizing the conformational flexibility of beta(2)-microglobulin amyloid fibrils at single-residue resolution that uses H/D exchange of amide protons combined with NMR analysis. The results indicate that most residues in the middle region of the molecule, including the loop regions in the native structure, form a rigid beta-sheet core, whereas the the N- and C-termini are excluded from this core. The extensively hydrogen-bonded beta-sheet core explains the remarkable rigidity and stability of amyloid fibrils. The present method could be used to obtain residue-specific conformational information of various amyloid fibrils, even though it does not provide a high resolution three-dimensional structure.     

Conformational dynamics of beta(2)-microglobulin analyzed by reduction and reoxidation of the disulfide bond

Although native beta(2)-microglobulin (beta2-m), the light chain of the major histocompatibility complex class I antigen, assumes an immunoglobulin domain fold, it is also found as a major component of dialysis-related amyloid fibrils. In the amyloid fibrils, the conformation of beta2-m is considered to be largely different from that of the native state, and a monomeric denatured form is likely to be a precursor to the amyloid fibril. To obtain insight into the conformational dynamics of beta2-m leading to the formation of amyloid fibrils, we studied the reduction and reoxidation of the disulfide bond by reduced and oxidized dithiothreitol, respectively, and the effects on the reduction of the chaperonin GroEL, a model protein that might destabilize the native state of beta2-m. We show that beta2-m occasionally unfolds into a denatured form even under physiological conditions and that this transition is promoted upon interaction with GroEL. The results imply that in vivo interactions of beta2-m with other proteins or membrane components could destabilize its native structure, thus stabilizing the amyloid precursor.     

Structural polymorphism and possible pathways of amyloid fibril formation on the example of insulin protein

In this review we analyze the main works on amyloid formation of insulin. There are many environmental factors affecting the formation of insulin amyloid fibrils (and other amyloidogenic proteins) such as: protein concentration, pH, ionic strength of solution, medium composition (anions, cations), presence of denaturants (urea, guanidine chloride) or stabilizers (saccharose), temperature regime, agitation. Since polymorphism is potentially crucial for human diseases and may underlie the natural variability of some amyloid diseases, in this review we focus attention on polymorphism that is an important biophysical difference between native protein folding suggesting correspondence between the amino acid sequence and unique folding state, and formation of amyloid fibrils, when the same amino acid sequence can form amyloid fibrils of different morphology. At present, according to the literature data, we can choose three ways of polymerization of insulin molecules depending on the nucleus size. The first suggests that fibrillogenesis can occur through assembly of insulin monomers. The second suggests that precursors of fibrils are dimers, and the third assumes that precursors of fibrils are oligomers. Additional experimental works and new methods of investigation and assessment of results are needed to clarify the general picture of insulin amyloid formation.     

Characterization of two distinct beta2-microglobulin unfolding intermediates that may lead to amyloid fibrils of different morphology

The self-assembly of beta(2)-microglobulin into fibrils leads to dialysis-related amyloidosis. pH-mediated partial unfolding is required for the formation of the amyloidogenic intermediate that then self-assembles into amyloid fibrils. Two partially folded intermediates of beta(2)-microglobulin have been identified experimentally and linked to the formation of fibrils of distinct morphology, yet it remains difficult to characterize these partially unfolded states at high resolution using experimental approaches. Consequently, we have performed molecular dynamics simulations at neutral and low pH to determine the structures of these partially unfolded amyloidogenic intermediates. In the low-pH simulations, we observed the formation of alpha-sheet structure, which was first proposed by Pauling and Corey. Multiple simulations were performed, and two distinct intermediate state ensembles were identified that may account for the different fibril morphologies. The predominant early unfolding intermediate was nativelike in structure, in agreement with previous NMR studies. The late unfolding intermediate was significantly disordered, but it maintained an extended elongated structure, with hydrophobic clusters and residual alpha-extended chain strands in specific regions of the sequence that map to amyloidogenic peptides. We propose that the formation of alpha-sheet facilitates self-assembly into partially unfolded prefibrillar amyloidogenic intermediates.     

Progress towards a molecular-level structural understanding of amyloid fibrils

The problem of determining and understanding the molecular structures of amyloid fibrils has attracted considerable attention and effort over the past several years. Although complete, high-resolution structures have not yet been obtained, key features of protein and peptide conformations and supramolecular organization within amyloid fibrils have been elucidated using a variety of novel experimental methods, including magnetic resonance spectroscopies, electron microscopy, X-ray and neutron scattering, and biochemical techniques. The experimental data are beginning to shed light on issues such as the nature of the intermolecular interactions that stabilize amyloid structures, the molecular structural basis for polymorphism in amyloid fibrils, the universality of amyloid structures, and the balance between structural order and disorder within amyloid fibrils. Recent structural data will contribute to an improved understanding of the mechanisms of amyloid formation and to the development of therapeutic agents for amyloid diseases.     

Heat-triggered conversion of protofibrils into mature amyloid fibrils of beta2-microglobulin

Heat-triggered conversion of the salt-induced thin and flexible protofibrils into well-organized thick and straight mature amyloid fibrils was achieved with beta2-microglobulin, a protein responsible for dialysis-related amyloidosis. First, protofibrils that formed spontaneously at pH 2.5 in the presence of 0.5 M NaCl were aggregated by agitating the solution. Second, the aggregated protofibrils were heated in a cell of a differential scanning calorimeter (DSC). The DSC thermogram showed an exothermic transition with sigmoidal temperature dependence, resulting in a remarkably large decrease in the heat capacity of the solution. Third, on the basis of electron microscopy together with circular dichroism spectroscopy, seeding experiments, and a thioflavin T binding assay, the sigmoidal transition was found to represent the conversion of protofibrils into mature amyloid fibrils. Furthermore, DSC thermograms obtained at various heating rates revealed that the transition curve depends on the heating rate, implying that the effects of heat associated with the conversion to the mature fibrils are kinetically controlled, precluding an interpretation in terms of equilibrium thermodynamics. Taken together, these results highlight the importance of the change in heat capacity in addressing the biological significance of interactions between solvent water and amyloid fibrils and, moreover, in detecting the formation of amyloid fibrils.