Role of the single disulphide bond of beta(2)-microglobulin in amyloidosis in vitro

The aggregation of beta(2)-microglobulin (beta(2)m) into amyloid fibrils occurs in the condition known as dialysis-related amyloidosis (DRA). The protein has a beta-sandwich fold typical of the immunoglobulin family, which is stabilized by a highly conserved disulphide bond linking Cys25 and Cys80. Oxidized beta(2)m forms amyloid fibrils rapidly in vitro at acidic pH and high ionic strength. Here we investigate the role of the single disulphide bond of beta(2)m in amyloidosis in vitro. We show that reduction of the disulphide bond destabilizes the native protein such that non-native molecules are populated at neutral pH. These species are prone to oligomerization but do not form amyloid fibrils when incubated for up to 8 mo at pH 7.0 in 0.4 M NaCl. Over the pH range 4.0-1.5 in the presence of 0.4 M NaCl, however, amyloid fibrils of reduced beta(2)m are formed. These fibrils are approximately 10 nm wide, but are shorter and assemble more rapidly than those produced from the oxidized protein. These data show that population of non-native conformers of beta(2)m at neutral pH by reduction of its single disulphide bond is not sufficient for amyloid formation. Instead, association of one or more specific partially unfolded molecules formed at acid pH are necessary for the formation of beta(2)m amyloid in vitro. Further experiments will now be needed to determine the role of different oligomeric species of beta(2)m in the toxicity of the protein in vivo.     

A systematic study of the effect of physiological factors on beta2-microglobulin amyloid formation at neutral pH

Beta(2)-microglobulin (beta(2)m) forms amyloid fibrils that deposit in the musculo-skeletal system in patients undergoing long-term hemodialysis. How beta(2)m self-assembles in vivo is not understood, since the monomeric wild-type protein is incapable of forming fibrils in isolation in vitro at neutral pH, while elongation of fibril-seeds made from recombinant protein has only been achieved at low pH or at neutral pH in the presence of detergents or cosolvents. Here we describe a systematic study of the effect of 11 physiologically relevant factors on beta(2)m fibrillogenesis at pH 7.0 without denaturants. By comparing the results obtained for the wild-type protein with those of two variants (DeltaN6 and V37A), the role of protein stability in fibrillogenesis is explored. We show that DeltaN6 forms low yields of amyloid-like fibrils at pH 7.0 in the absence of seeds, suggesting that this species could initiate fibrillogenesis in vivo. By contrast, high yields of amyloid-like fibrils are observed for all proteins when assembly is seeded with fibril-seeds formed from recombinant protein at pH 2.5 stabilized by the addition of heparin, serum amyloid P component (SAP), apolipoprotein E (apoE), uremic serum, or synovial fluid. The results suggest that the conditions within the synovium facilitate fibrillogenesis of beta(2)m and show that different physiological factors may act synergistically to promote fibril formation. By comparing the behavior of wild-type beta(2)m with that of DeltaN6 and V37A, we show that the physiologically relevant factors enhance fibrillogenesis by stabilizing fibril-seeds, thereby allowing fibril extension by rare assembly competent species formed by local unfolding of native monomers.     

Amyloid-forming peptides from beta2-microglobulin-Insights into the mechanism of fibril formation in vitro

Beta(2)-Microglobulin (beta(2)m) is one of over 20 proteins known to be involved in human amyloid disease. Peptides equivalent to each of the seven beta-strands of the native protein, together with an eighth peptide (corresponding to the most stable region in the amyloid precursor conformation formed at pH 3.6, that includes residues in the native strand E plus the eight succeeding residues (named peptide E')), were synthesised and their ability to form fibrils investigated. Surprisingly, only two sequences, both of which encompass the region that forms strand E in native beta(2)m, are capable of forming amyloid-like fibrils in vitro. These peptides correspond to residues 59-71 (peptide E) and 59-79 (peptide E') of intact beta(2)m. The peptides form fibrils under the acidic conditions shown previously to promote amyloid formation from the intact protein (pH <5 at low and high ionic strength), and also associate to form fibrils at neutral pH. Fibrils formed from these two peptides enhance fibrillogenesis of the intact protein. No correlation was found between secondary structure propensity, peptide length, pI or hydrophobicity and the ability of the peptides to associate into amyloid-like fibrils. However, the presence of a relatively high content of aromatic side-chains correlates with the ability of the peptides to form amyloid fibrils. On the basis of these results we propose that residues 59-71 may be important in the self-association of partially folded beta(2)m into amyloid fibrils and discuss the relevance of these results for the assembly mechanism of the intact protein in vitro.     

Role of the N and C-terminal strands of beta 2-microglobulin in amyloid formation at neutral pH

Beta 2-microglobulin (beta(2)m) is known to form amyloid fibrils de novo in vitro under acidic conditions (below pH 4.8). Fibril formation at neutral pH, however, has only been observed by deletion of the N-terminal six residues; by the addition of pre-assembled seeds; or in the presence of Cu(2+). Based on these observations, and other structural data, models for fibril formation of beta(2)m have been proposed that involve the fraying of the N and C-terminal beta-strands and the consequent loss of edge strand protective features. Here, we examine the role of the N and C-terminal strands in the initiation of fibrillogenesis of beta(2)m by creating point mutations in strands A and G and comparing the properties of the resulting proteins with variants containing similar mutations elsewhere in the protein. We show that truncation of buried hydrophobic side-chains in strands A and G promotes rapid fibril formation at neutral pH, even in unseeded reactions, and increases the rate of fibril formation under acidic conditions. By contrast, similar mutations created in the remaining seven beta-strands of the native protein have little effect on the rate or pH dependence of fibril formation. The data are consistent with the view that perturbation of the N and C-terminal edge strands is an important feature in the generation of assembly-competent states of beta(2)m.     

Production and characterization of RNA aptamers specific for amyloid fibril epitopes

One of the most fascinating features of amyloid fibrils is their generic cross-beta architecture that can be formed from many different and completely unrelated proteins. Nonetheless, amyloid fibrils with diverse structural and phenotypic properties can form, both in vivo and in vitro, from the same protein sequence. Here, we have exploited the power of RNA selection techniques to isolate small, structured, single-stranded RNA molecules known as aptamers that were targeted specifically to amyloid-like fibrils formed in vitro from beta(2)-microglobulin (beta(2)m), the amyloid fibril protein associated with dialysis-related amyloidosis. The aptamers bind with high affinity (apparent K(D) approximately nm) to beta(2)m fibrils with diverse morphologies generated under different conditions in vitro, as well as to amyloid fibrils isolated from tissues of dialysis-related amyloidosis patients, demonstrating that they can detect conserved epitopes between different fibrillar species of beta(2)m. Interestingly, the aptamers also recognize some other, but not all, amyloid fibrils generated in vitro or isolated from ex vivo sources. Based on these observations, we have shown that although amyloid fibrils share many common structural properties, they also have features that are unique to individual fibril types.     

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.     

Amyloid fibril formation in the context of full-length protein: effects of proline mutations on the amyloid fibril formation of beta2-microglobulin

Beta2-microglobulin (beta2-m), a typical immunoglobulin domain made of seven beta-strands, is a major component of amyloid fibrils formed in dialysis-related amyloidosis. To understand the mechanism of amyloid fibril formation in the context of full-length protein, we prepared various mutants in which proline (Pro) was introduced to each of the seven beta-strands of beta2-m. The mutations affected the amyloidogenic potential of beta2-m to various degrees. In particular, the L23P, H51P, and V82P mutations significantly retarded fibril extension at pH 2.5. Among these, only L23P is included in the known "minimal" peptide sequence, which can form amyloid fibrils when isolated as a short peptide. This indicates that the residues in regions other than the minimal sequence, such as H51P and V82P, determine the amyloidogenic potential in the full-length protein. To further clarify the mutational effects, we measured their stability against guanidine hydrochloride of the native state at pH 8.0 and the amyloid fibrils at pH 2.5. The amyloidogenicity of mutants showed a significant correlation with the stability of the amyloid fibrils, and little correlation was observed with that of the native state. It has been proposed that the stability of the native state and the unfolding rate to the amyloidogenic precursor as well as the conformational preference of the denatured state determine the amyloidogenicity of the proteins. The present results reveal that, in addition, stability of the amyloid fibrils is a key factor determining the amyloidogenic potential of the proteins.     

Theophylline-induced apoptosis is paralleled by protein kinase A-dependent tissue transglutaminase activation in cancer cells

beta(2)-Microglobulin (beta2M), the light chain of the type I major histocompatibility complex, is a major component of dialysis-related amyloid fibrils. beta2M in the native state has a typical immunoglobulin fold with a buried intrachain disulfide bond. The conformation and stability of recombinant beta2M in which the intrachain disulfide bond was reduced were studied by CD, tryptophan fluorescence, and one-dimensional NMR. The conformation of the reduced beta2M in the absence of denaturant at pH 8.5 was similar to that of the intact protein unless the thiol groups were modified. However, reduction of the disulfide bond decreased the stability as measured by denaturation in guanidine hydrochloride. Intact beta2M formed amyloid fibrils at pH 2.5 by extension reaction using sonicated amyloid fibrils as seeds. Under the same conditions, reduced beta2M did not form typical amyloid fibrils, although it inhibited fibril extension competitively, suggesting that the conformation defined by the disulfide bond is important for amyloid fibril formation of beta2M.     

Hierarchical assembly of beta2-microglobulin amyloid in vitro revealed by atomic force microscopy

The kinetics of spontaneous assembly of amyloid fibrils of wild-type beta(2)-microglobulin (beta(2)M) in vitro, under acid conditions (pH 2.5) and low ionic strength, has been followed using thioflavin-T (ThT) binding. In parallel experiments, the morphology of the different fibrillar species present at different time-points during the growth process were characterised using tapping-mode atomic force microscopy (TM-AFM) in air and negative stain electron microscopy (EM). The thioflavin-T assay shows a characteristic lag phase during which the nucleation of fibrils occurs before a rapid growth in fibril density. The volume of fibrils deposited on mica measured from TM-AFM images at each time-point correlates well with the fluorescence data. TM-AFM and negative-stain EM revealed the presence of various kinds of protein aggregates in the lag phase that disappear concomitantly with a rise in the density of amyloid fibrils, suggesting that these aggregates precede fibril growth and may act as nucleation sites. Three distinct morphologies of mature amyloid fibrils were observed within a single growth experiment, as observed previously for the wild-type protein and the variant N17D. Additional supercoiled morphologies of the lower-order fibrils were observed. Comparative height analysis from the TM-AFM data allows each of the mature fibril types and single protofilaments to be identified unambiguously, and reveals that the assembly occurs via a hierarchy of morphological states.     

Structure of interacting segments in the growing amyloid fibril of beta2-microglobulin probed with IR spectroscopy

Inter-segmental interaction at the growing tip of the amyloid fibril of beta2-microglobulin (beta2m) was investigated using IR microscopy. Cross-seeded fibril formation was implemented, in which the amyloid fibril of the #21-31 fragment of beta2m (fA[#21-31]) was generated on the beta2m amyloid fibril (fA[beta2m]) as a seed. Differences between the IR spectra of the cross-seeded fibril and those of the seed were attributed to the contribution from the tip, whose structure is discussed. The results indicated that 6.5 +/- 1.0 out of 11 residues of the fA[#21-31] tip on fA[beta2m] are contained in a beta-sheet at pH 2.5, which was smaller than the corresponding value (7.5 +/- 1.1 residues) of the spontaneous fA[#21-31] at pH 2.5. The tip was suggested to have a planar structure, indicating the planarity of the interacting segment. The N-terminal region of fA[#21-31] in the fibril is more exposed to the solvent than that in the tip, and vice versa for the C-terminal region. This is consistent with the different protonation levels of these regions, and the direction of peptide in the fibrils is determined from these results.     

Core and heterogeneity of beta2-microglobulin amyloid fibrils as revealed by H/D exchange

Dialysis-related amyloidosis, which occurs in the patients receiving a long-term hemodialysis with high frequency, accompanies the deposition of amyloid fibrils composed of beta(2)-microglobulin (beta2-m). In vitro, beta2-m forms two kinds of fibrous structures at acidic pH. One is a rigid "mature fibril", and the other is a flexible thin filament often called an "immature fibril". In addition, a 22-residue peptide (K3 peptide) corresponding to Ser20 to Lys41 of intact beta2-m forms rigid amyloid-like fibrils similar to mature fibrils. We compared the core of these three fibrils at single-residue resolution using a recently developed hydrogen/deuterium (H/D) exchange method with the dissolution of fibrils by dimethylsulfoxide (DMSO). The exchange time-course of these fibrils showed large deviations from a single exponential curve showing that, because of the supramolecular structures, the same residue exists in different environments from molecule to molecule, even in a single fibril. The exchange profiles revealed that the core of the immature fibril is restricted to a narrow region compared to that of the mature fibril. In contrast, all residues were protected from exchange in the K3 fibril, indicating that a whole region of the peptide is engaged in the beta-sheet network. These results suggest the mechanism of amyloid fibril formation, in which the core beta-sheet formed by a minimal sequence propagates to form a rigid and extensive beta-sheet network.     

Co-populated conformational ensembles of beta2-microglobulin uncovered quantitatively by electrospray ionization mass spectrometry

Ordered assembly of monomeric human beta(2)-microglobulin (beta(2)m) into amyloid fibrils is associated with the disorder hemodialysis-related amyloidosis. Previously, we have shown that under acidic conditions (pH <5.0 at 37 degrees C), wild-type beta(2)m assembles spontaneously into fibrils with different morphologies. Under these conditions, beta(2)m populates a number of different conformational states in vitro. However, this equilibrium mixture of conformationally different species is difficult to resolve using ensemble techniques such as nuclear magnetic resonance or circular dichroism. Here we use electrospray ionization mass spectrometry to resolve different species of beta(2)m populated between pH 6.0 and 2.0. We show that by linear deconvolution of the charge state distributions, the extent to which each conformational ensemble is populated throughout the pH range can be determined and quantified. Thus, at pH 3.6, conditions under which short fibrils are produced, the conformational ensemble is dominated by a charge state distribution centered on the 9+ ions. By contrast, under more acidic conditions (pH 2.6), where long straight fibrils are formed, the charge state distribution is dominated by the 10+ and 11+ ions. The data are reinforced by investigations on two variants of beta(2)m (V9A and F30A) that have reduced stability to pH denaturation and show changes in the pH dependence of the charge state distribution that correlate with the decrease in stability measured by tryptophan fluorescence. The data highlight the potential of electrospray ionization mass spectrometry to resolve and quantify complex mixtures of different conformational species, one or more of which may be important in the formation of amyloid.     

Partially unfolded states of beta(2)-microglobulin and amyloid formation in vitro

Dialysis-related amyloidosis (DRA) involves the aggregation of beta(2)-microglobulin (beta(2)m) into amyloid fibrils. Using Congo red and thioflavin-T binding, electron microscopy, and X-ray fiber diffraction, we have determined conditions under which recombinant monomeric beta(2)m spontaneously associates to form fibrils in vitro. Fibrillogenesis is critically dependent on the pH and the ionic strength of the solution, with low pH and high ionic strength favoring fibril formation. The morphology of the fibrils formed varies with the growth conditions. At pH 4 in 0.4 M NaCl the fibrils are approximately 10 nm wide, relatively short (50-200 nm), and curvilinear. By contrast, at pH 1.6 the fibrils formed have the same width and morphology as those formed at pH 4 but extend to more than 600 nm in length. The dependence of fibril growth on ionic strength has allowed the conformational properties of monomeric beta(2)m to be determined under conditions where fibril growth is impaired. Circular dichroism studies show that titration of one or more residues with a pK(a) of 4.7 destabilizes native beta(2)m and generates a partially unfolded species. On average, these molecules retain significant secondary structure and have residual, non-native tertiary structure. They also bind the hydrophobic dye 1-anilinonaphthalene-8-sulfonic acid (ANS), show line broadening in one-dimensional (1)H NMR spectra, and are weakly protected from hydrogen exchange. Further acidification destabilizes this species, generating a second, more highly denatured state that is less fibrillogenic. These data are consistent with a model for beta(2)m fibrillogenesis in vitro involving the association of partially unfolded molecules into ordered fibrillar assemblies.     

Ultrasonication-induced amyloid fibril formation of beta2-microglobulin

To obtain insight into the mechanism of fibril formation, we examined the effects of ultrasonication, a strong agitator, on beta2-microglobulin (beta2-m), a protein responsible for dialysis-related amyloidosis. Upon sonication of an acid-unfolded beta2-m solution at pH 2.5, thioflavin T fluorescence increased markedly after a lag time of 1-2 h with a simultaneous increase of light scattering. Atomic force microscopy images showed the formation of a large number of short fibrils 3 nm in diameter. When the sonication-induced fibrils were used as seeds in the next seeding experiment at pH 2.5, a rapid and intense formation of long fibrils 3 nm in diameter was observed demonstrating seed-dependent fibril growth. We then examined the effects of sonication on the native beta2-m at neutral pH, conditions under which amyloid deposits occur in patients. In the presence of 0.5 mm sodium dodecyl sulfate, a model compound of potential trigger and stabilizer of amyloid fibrils in patients, a marked increase of thioflavin T fluorescence was observed after 1 day of sonication at pH 7.0. The products of sonication caused the accelerated fibril formation at pH 7.0. Atomic force microscopy images showed that the fibrils formed at pH 7.0 have a diameter of more than 7 nm, thicker than those prepared at pH 2.5. These results indicate that ultrasonication is one form of agitation triggering the formation of amyloid fibrils of beta2-m, producing fibrils adapted to the respective pH.     

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.     

Investigation into the role of macrophages in the formation and degradation of beta2-microglobulin amyloid fibrils

Dialysis related amyloidosis is a serious complication of long-term hemodialysis in which beta(2)-microglobulin (beta(2)m) forms amyloid fibrils that deposit predominantly in cartilaginous tissues. How these fibrils form in vivo, however, is poorly understood. Here we perform a systematic investigation into the role of macrophages in the formation and degradation of beta(2)m amyloid fibrils, building on observations that macrophages are found in association with beta(2)m amyloid deposits in vivo and that these cells contain intra-lysosomal beta(2)m amyloid. In live cell imaging experiments we demonstrate that macrophages internalize monomeric beta(2)m, whereupon it is sorted to lysosomes. At lysosomal pH beta(2)m self-associates in vitro to form amyloid-like fibrils with an array of morphologies as visualized by atomic force microscopy. Cleavage of the monomeric protein by both macrophages and lysosomal proteases isolated from these cells results in the rapid degradation of the monomeric protein, preventing amyloid formation. Incubation of macrophages with preformed fibrils revealed that macrophages internalize amyloid-like fibrils formed extracellularly, but in marked contrast with the monomeric protein, the fibrils were not degraded within macrophage lysosomes. Correspondingly beta(2)m fibrils were highly resistant to degradation by high concentrations of lysosomal proteases isolated from macrophages. Despite their enormous degradative capacity, therefore, macrophage lysosomes cannot ameliorate dialysis-related amyloidosis by degrading pre-existing amyloid fibrils, but lysosomal proteases may play a protective role by eliminating amyloid precursors before beta(2)m fibrils can accumulate in what may represent an otherwise fibrillogenic environment.     

Strong acids induce amyloid fibril formation of β2-microglobulin via an anion-binding mechanism

Amyloid fibrils, crystal-like fibrillar aggregates of proteins associated with various amyloidoses, have the potential to propagate via a prion-like mechanism. Among known methodologies to dissolve preformed amyloid fibrils, acid treatment has been used with the expectation that the acids will degrade amyloid fibrils similar to acid inactivation of protein functions. Contrary to our expectation, treatment with strong acids, such as HCl or H₂SO₄, of β₂-microglobulin (β2m) or insulin actually promoted amyloid fibril formation, proportionally to the concentration of acid used. A similar promotion was observed at pH 2.0 upon the addition of salts, such as NaCl or Na₂SO₄. Although trichloroacetic acid, another strong acid, promoted amyloid fibril formation of β2m, formic acid, a weak acid, did not, suggesting the dominant role of anions in promoting fibril formation of this protein. Comparison of the effects of acids and salts confirmed the critical role of anions, indicating that strong acids likely induce amyloid fibril formation via an anion-binding mechanism. The results suggest that although the addition of strong acids decreases pH, it is not useful for degrading amyloid fibrils, but rather induces or stabilizes amyloid fibrils via an anion-binding mechanism.     

Competing pathways determine fibril morphology in the self-assembly of beta2-microglobulin into amyloid

Despite its importance in biological phenomena, a comprehensive understanding of the mechanism of amyloid formation remains elusive. Here, we use atomic force microscopy to map the formation of beta2-microglobulin amyloid fibrils with distinct morphologies and persistence lengths, when protein concentration, pH and ionic strength are varied. Using the resulting state-diagrams, we demonstrate the existence of two distinct competitive pathways of assembly, which define an energy landscape that rationalises the sensitivity of fibril morphology on the solution conditions. Importantly, we show that semi-flexible (worm-like) fibrils, which form rapidly during assembly, are kinetically trapped species, formed via a non-nucleated pathway that is explicitly distinct from that leading to the formation of the relatively rigid long-straight fibrils classically associated with amyloid. These semi-flexible fibrils also share an antibody epitope common to other protein oligomers that are known to be toxic species linked to human disease. The results demonstrate the heterogeneity of amyloid assembly, and have important implications for our understanding of the importance of oligomeric states in amyloid disease, the origins of prion strains, and the development of therapeutic strategies.     

Thermodynamic modulation of light chain amyloid fibril formation

To obtain further insight into the pathogenesis of amyloidosis and develop therapeutic strategies to inhibit fibril formation we investigated: 1) the relationship between intrinsic physical properties (thermodynamic stability and hydrogen-deuterium (H-D) exchange rates) and the propensity of human immunoglobulin light chains to form amyloid fibrils in vitro; and 2) the effects of extrinsically modulating these properties on fibril formation. An amyloid-associated protein readily formed amyloid fibrils in vitro and had a lower free energy of unfolding than a homologous nonpathological protein, which did not form fibrils in vitro. H-D exchange was much faster for the pathological protein, suggesting it had a greater fraction of partially folded molecules. The thermodynamic stabilizer sucrose completely inhibited fibril formation by the pathological protein and shifted the values for its physical parameters to those measured for the nonpathological protein in buffer alone. Conversely, urea sufficiently destabilized the nonpathological protein such that its measured physical properties were equivalent to those of the pathological protein in buffer, and it formed fibrils. Thus, fibril formation by light chains is predominantly controlled by thermodynamic stability; and a rational strategy to inhibit amyloidosis is to design high affinity ligands that specifically increase the stability of the native protein.