Supramolecular ligands: Monomer structure and protein ligation capability

The aim of this work was to define the chemical structure of compounds self-assembling in water solutions, which appear to interact with proteins as single ligands with their supramolecular nature preserved. For this purpose the ligation to proteins of his azo dyes, represented by Congo red and its derivatives with designed structural alterations, were tested. The three parameters which characterize the reactivity of supramolecular material were determined in the same conditions for all studied dyes. These were: A) stability of the assembly products; B) binding to heat-denatured protein (human IgG); and C) binding to native protein (rabbit antibodies in the immune complex) measured by the enhancement of hemagglutination. The structural differences between the Congo red derivatives concerned the symmetry of the molecule and the structure of its non-polar component, which occupies the central part of the dye molecule and is thought to be crucial for self-assembly. Other dyes were also studied for the same purpose: Evans blue and Trypan blue, bis-ANS and ANS, as well as a group of compounds with a structural design unlike that of bis azo dyes. Compounds with rigid elongated symmetric molecules with a large non-polar middle fragment are expected to form a ribbon-like supramolecular organization in assembling. They appeared to have ligation properties related to their self-assembling tendency. The compounds with different structures, not corresponding to his azo dyes, did not reveal ligation capability, at least in respect to native protein. The conditions of binding to denatured proteins seem less restrictive than the conditions of binding to native molecules. The molten hydrophobic protein interior becomes a new binding area allowing for complexation of even non-assembled molecules.     

Is Congo red an amyloid-specific dye?

Congo red (CR) binding, monitored by characteristic yellow-green birefringence under crossed polarization has been used as a diagnostic test for the presence of amyloid in tissue sections for several decades. This assay is also widely used for the characterization of in vitro amyloid fibrils. In order to probe the structural specificity of Congo red binding to amyloid fibrils we have used an induced circular dichroism (CD) assay. Amyloid fibrils from insulin and the variable domain of Ig light chain demonstrate induced CD spectra upon binding to Congo red. Surprisingly, the native conformations of insulin and Ig light chain also induced Congo red circular dichroism, but with different spectral shapes than those from fibrils. In fact, a wide variety of native proteins exhibited induced CR circular dichroism indicating that CR bound to representative proteins from different classes of secondary structure such as alpha (citrate synthase), alpha + beta (lysozyme), beta (concavalin A), and parallel beta-helical proteins (pectate lyase). Partially folded intermediates of apomyoglobin induced different Congo red CD bands than the corresponding native conformation, however, no induced CD bands were observed with unfolded protein. Congo red was also found to induce oligomerization of native proteins, as demonstrated by covalent cross-linking and small angle x-ray scattering. Our data suggest that Congo red is sandwiched between two protein molecules causing protein oligomerization. The fact that Congo red binds to native, partially folded conformations and amyloid fibrils of several proteins shows that it must be used with caution as a diagnostic test for the presence of amyloid fibrils in vitro.     

Evidence that supramolecular Congo red is the sole ligation form of this dye for L chain lambda derived amyloid proteins.

The mechanism of Congo red binding to amyloid protein was studied in order to establish which of two structural dye versions present in water solutions--unimolecular and supramolecular--represent its actual ligation form. Immunoglobulin L chain lambda of amyloidogenic nature, expressed by Congo red binding and easy gel formation, was used as the model amyloid protein. Congo red was coassembled with rhodamine B, designed to be a marker of the Congo red micellar organisation in complexation with protein. The particular suitability of rhodamine B for this role results from significant difference in its binding affinity to Congo red and to protein. It associates readily with Congo red, becoming incorporated into its micellar organisation, but as homogenous dye it shows an almost complete inability to bind to protein. In view of these properties, Congo red was used as a vehicle to draw rhodamine B into complexation with protein, at the same time supplying evidence of its supramolecular ligation form. The results show that both soluble amyloid precursor L chain and the derived gel material attach rhodamine B coassembled with Congo red but not the homogenous rhodamine B. Despite its dynamic, supramolecular character, Congo red participates in complexation with amyloid proteins as an integral ligand unit.     

The structure and protein binding of amyloid-specific dye reagents

The self-assembling tendency and protein complexation capability of dyes related to Congo red and also some dyes of different structure were compared to explain the mechanism of Congo red binding and the reason for its specific affinity for beta-structure. Complexation with proteins was measured directly and expressed as the number of dye molecules bound to heat-aggregated IgG and to two light chains with different structural stability. Binding of dyes to rabbit antibodies was measured indirectly as the enhancement effect of the dye on immune complex formation. Self-assembling was tested using dynamic light scattering to measure the size of the supramolecular assemblies. In general the results show that the supramolecular form of a dye is the main factor determining its complexation capability. Dyes that in their compact supramolecular organization are ribbon-shaped may adhere to polypeptides of beta-conformation due to the architectural compatibility in this unique structural form. The optimal fit in complexation seems to depend on two contradictory factors involving, on the one hand, the compactness of the non-covalently stabilized supramolecular ligand, and the dynamic character producing its plasticity on the other. As a result, the highest protein binding capability is shown by dyes with a moderate self-assembling tendency, while those arranging into either very rigid or very unstable supramolecular entities are less able to bind.     

Sonication of proteins causes formation of aggregates that resemble amyloid

Despite the widespread use of sonication in medicine, industry, and research, the effects of sonication on proteins remain poorly characterized. We report that sonication of a range of structurally diverse proteins results in the formation of aggregates that have similarities to amyloid aggregates. The formation of amyloid is associated with, and has been implicated in, causing of a wide range of protein conformational disorders including Alzheimer's disease, Huntington's disease, Parkinson's disease, and prion diseases. The aggregates cause large enhancements in fluorescence of the dye thioflavin T, exhibit green-gold birefringence upon binding the dye Congo red, and cause a red-shift in the absorbance spectrum of Congo red. In addition, circular dichroism reveals that sonication-induced aggregates have high beta-content, and proteins with significant native alpha-helical structure show increased beta-structure in the aggregates. Ultrastructural analysis by electron microscopy reveals a range of morphologies for the sonication-induced aggregates, including fibrils with diameters of 5-20 nm. The addition of preformed aggregates to unsonicated protein solutions results in accelerated and enhanced formation of additional aggregates upon heating. The dye-binding and structural characteristics, as well as the ability of the sonication-induced aggregates to seed the formation of new aggregates are all similar to the properties of amyloid. These results have important implications for the use of sonication in food, biotechnological and medical applications, and for research on protein aggregation and conformational disorders.     

Supramolecularity creates nonstandard protein ligands

Congo red and a group of structurally related dyes long used to stain amyloid proteins are known to associate in water solutions. The self-association of some dyes belonging to this group appears particularly strong. In water solutions their molecules are arranged in ribbon-like micellar forms with liquid crystalline properties. These compounds have recently been found to form complexes with some native proteins in a non-standard way. Gaps formed by the local distribution of beta-sheets in proteins probably represent the receptor sites for these dye ligands. They may result from higher structural instability in unfolding conditions, but also may appear as long range cooperative fluctuations generated by ligand binding. Immunoglobulins G were chosen as model binding proteins to check the mechanism of binding of these dyes. The sites of structural changes generated by antigen binding in antibodies, believed to act as a signal propagated to distant parts of the molecule, were assumed to be suitable sites for the complexation of liquid-crystalline dyes. This assumption was confirmed by proving that antibodies engaged in immune complexation really do bind these dyes; as expected, this binding affects their function by significantly enhancing antigen binding and simultaneously inhibiting C1q attachment. Binding of these supramolecular dyes by some other native proteins including serpins and their natural complexes was also shown. The strict dependence of the ligation properties on strong self-assembling and the particular arrangement of dye molecules indicate that supramolecularity is the feature that creates non-standard protein ligands, with potential uses in medicine and experimental science.     

FT-Raman spectroscopy as diagnostic tool of Congo red binding to amyloids

Chorion is the major component of silkmoth eggshell. More than 95% of its dry mass consists of the A and B families of low molecular weight structural proteins, which have remarkable mechanical and chemical properties protecting the oocyte and developing embryo from environmental hazards. We present data from FT-Raman spectroscopy of silkmoth chorion and amyloid-like fibrils formed from peptide analogues of chorion proteins, both unstained and stained by Congo red. The results show that FT-Raman spectroscopy is not a straightforward diagnostic tool for the specific interactions of Congo red with amyloids: a dilute aqueous solution of the Congo red dye at pH 5.5 and a thin solid film of the dye cast from this solution exhibit the same "diagnostic" Raman shifts relative to the neat Congo red dry powder as do amyloid fibrils formed from peptide analogues of chorion proteins stained by Congo red. An important consequence of this finding is that these shifts of the Raman active modes of Congo red are probably due to the formation of supramolecular dye aggregates in the presence of water. Therefore, this is not an appropriate diagnostic test for Congo red binding to amyloids.     

Amyloid from a histochemical perspective. A review of the structure,properties and types of amyloid,and a proposed staining mechanism for Congo red staining

Amyloid is a diverse group of unrelated peptides or proteins that have positive functionality or are associated with various pathologies. Despite vast differences, all amyloids share several features that together uniquely define the group. 1) All amyloids possess a characteristic cross-ß pattern with X-ray diffraction typical of ß-sheet secondary protein structures. 2) All amyloids are birefringent and dichroic under polarizing microscopy after staining with Congo red, which indicates a crystalline-like (ordered) structure. 3) All amyloids cause a spectral shift in the peak wavelength of Congo red with conventional light microscopy due to perturbation of π electrons of the dye. 4) All amyloids show heightened intensity of fluorescence with Congo red, which suggests an unusual degree of packing of the dye onto the substrate. The ß portion of amyloid molecules, the only logical substrate for specific Congo red staining under histochemical conditions, consists of a stack of ß-sheets laminated by hydrophilic and hydrophobic interactions between adjacent pairs. Only the first and last ß-sheets are accessible to dyes. Each sheet is composed of numerous identical peptides running across the width of the sheet and arranged in parallel with side chains in register over the length of the fibril. Two sets of grooves are bordered by side chains. X grooves run perpendicular to the long axis of the fibril; these grooves are short (the width of the sheet) and number in the hundreds or thousands. Y grooves are parallel with the long axis. Each groove runs the entire length of the fibril, but there are very few of them. While Congo red is capable of ionic bonding with proteins via two sulfonic acid groups, physical constraints on the staining solution preclude ionic interactions. Hydrogen bonding between dye amine groups and peptide carbonyls is the most likely primary bonding mechanism, because all ß-sheets possess backbone carbonyls. Various amino acid residues may form secondary bonds to the dye via any of three van der Waals forces. It is possible that Congo red binds within the Y grooves, but that would not produce the characteristic staining features that are the diagnostic hallmarks of amyloid. Binding in the X grooves would produce a tightly packed series of dye molecules over the entire length of the fibril. This would account for the signature staining of amyloid by Congo red: dichroic birefringence, enhanced intensity of fluorescence and a shift in visible absorption wavelength.     

Binding of the dye congo red to the amyloid protein pig insulin reveals a novel homology amongst amyloid-forming peptide sequences.

The three-dimensional structure has been determined of a complex of the dye Congo Red, a specific stain for amyloid deposits, bound to the amyloid protein insulin. One dye molecule intercalates between two globular insulin molecules at an interface formed by a pair of anti-parallel beta-strands. This result, together with analysis of the primary sequences of other amyloidogenic proteins and peptides suggests that this mode of dye-binding to amyloid could be general. Moreover, the structure of this dye-binding interface between protein molecules provides an insight into the polymerization of amyloidogenic proteins into amyloid fibres. Thus the detailed characterization, at a resolution of 2.5 A, of the dye binding site in insulin could form a basis for the design of agents targeted against a variety of amyloid deposits.     

Influence of the electric field on supramolecular structure and properties of amyloid-specific reagent Congo red

Among specific amyloid ligands, Congo red and its analogues are often considered potential therapeutic compounds. However, the results of the studies so far have not been univocal because the properties of this dye, derived mostly from its supramolecular nature, are still poorly understood. The supramolecular structure of Congo red, formed by π-π stacking of dye molecules, is susceptible to the influence of the electric field, which may significantly facilitate electron delocalization. Consequently, the electric field may generate altered physico-chemical properties of the dye. Enhanced electron delocalization, induced by the electric field, alters the total charge of Congo red, making the dye more acidic (negatively charged). This is a consequence of withdrawing electrons from polar substituents of aromatic rings-sulfonic and amino groups-thus increasing their tendency to dissociate protons. The electric field-induced charge alteration observed in electrophoresis depends on dye concentration. This concentration-dependent charge alteration effect disappears when the supramolecular structure disintegrates in DMSO. Dipoles formed from supramolecular fibrillar species in the electric field become ordered in the solution, introducing the modified arrangement to liquid crystalline phase. Experimental results and theoretical studies provide evidence confirming predictions that the supramolecular character of Congo red is the main reason for its specific properties and reactivity.     

The cause of the green polarization color of amyloid stained with Congo red

Summary Experiments done with Congo red crystals and with Congo red deposits polished in a single direction by a glass wheel have shown that the appearance of green polarization color primarily depends on near-perfect parallel alignment of the dye particles. The green polarization color was seen only in the deposits which showed a clear transition from red to colorless when examined for dichroism. Another factor was found to be the thickness of the object, as the green polarization color was not present in too thick or too thin sections of amyloid-containing tissues stained with Congo red.The phenomena can be explained by the assumption that the green polarization color is due to interference between the red ray and the red component of the white ray whenever the retardation by the object approximates half the wavelength of red light.The findings indicate that amyloid differs from other materials which are stained by Congo red in that amyloid deposits bind the dye molecules in a more orderly and parallel fashion. It is suggested that minimal amounts of amyloid which are not visible in Congo red stained sections with ordinary light microscopy and which do not give the green polarization color can best be detected by examination for dichroism in ultraviolet light after having been stained with fluorescent dyes.     

Amyloid formation of native folded protein induced by peptide-based graft copolymer

We report here that a native folded holo-myoglobin, when incubated with a synthetic amyloidogenic peptide in aqueous solutions, forms fibrils. These fibrils took a cross-beta form (inter-strand spacing: 4.65 A and inter-sheet spacing: 10.65 A) and bound the amyloidophilic dye Congo red as did the authentic amyloid fibrils. In contrast such fibril formation of myoglobin did not occur in the absence of the peptide. These results suggest the possibility that inter-molecular interaction of native protein with the amyloidogenic peptide trigger the amyloid formation even for the non-pathogenic native protein like myoglobin, which itself exists as a globular form, under certain conditions.     

An approach to understand the complexation of supramolecular dye Congo red with immunoglobulin L chain lambda

Congo red, a dye of high self-assembling tendency, has been found to form complexes with proteins by adhesion of the ribbon-like supramolecular ligand to polypeptide chains of beta-conformation. Complexation is allowed by local or global protein instability, facilitating penetration of the dye to the locus of its binding. At elevated temperatures, L chain lambda of myeloma origin was found to form two distinct complexes with Congo red, easily differentiated in electrophoresis as slow- and fast-migrating fractions, bearing four- and eight-dye-molecule ligands, respectively, in the V domain of each individual chain. The slow-migrating complex is formed after displacement of the N-terminal polypeptide chain fragment (about 20 residues) from its packing locus, thereby exposing the entrance to the binding cavity. In this work the formation and stability of this complex was studied by molecular dynamics (MD) simulations. The effect of three- and five-molecule ligands introduced to the site binding the dye was also analyzed in an attempt to understand the formation of fast-migrating complexes. The wedging of the ligand containing five dye molecules, hence longer than established experimentally as the maximum for the slow-migrating complex, was found to generate significant structural changes. These changes were assumed to represent the crossing of the threshold on the way to forming a fast-migrating complex more capacious for dyes. They led to almost general destabilization of the V domain, making it susceptible to extra dye complexation. Theoretical studies were designed in close reference to experimental findings concerning the number of dye molecules in the ligand inserted to the site binding the dye, the location of the site in the domain, and the conditions of formation of the complexes. The results of the two kinds of studies appeared coherent.     

Methods for staining amyloid in tissues: a review

The traditional way of identifying amyloid in tissue sections has been staining with Congo red and demonstration of green birefringence under crossed polarizers. The original method of Congo red staining, described by Bennhold in 1922, has undergone several modifications to improve its sensitivity, specificity, and reliability. The most common modification is the alkaline Congo red method described by Puchtler and co-workers in 1962. Specificity is improved by using freshly prepared stain and a staining solution fully saturated with sodium chloride. Amyloid proteins can be further distinguished by autoclaving or by treating the tissue with potassium permanganate or alkaline guanidine. Autoclaving the tissues at 120 C for 30 min causes protein AA to lose its affinity for Congo red. Prolongation of autoclaving to 120 min abolishes the Congophilia of protein AL, but prealbumin-related amyloid shows little or no change. Treatment of the tissue with potassium permanganate causes protein AA and B2-microglobulin amyloid to lose their affinity to Congo red. Protein AA fails to stain with Congo red after treatment with alkaline guanidine for 1 min and protein AL and systemic senile amyloid protein (SSA) after 2 hr. Familial amyloid protein (FAP), prealbumin type, can stand 2 hr of alkaline guanidine treatment without losing its ability to stain with Congo red. Other methods of detection of amyloid include fluorescent stains, e.g., thioflavin T or S, and metachromatic stains such as crystal violet. Immunofluorescence and immunoperoxidase methods are used to identify and classify amyloid proteins in tissues. Antibodies against the P component, proteins AA and AL and FAP have been used with great precision. Due to cross-reactivity, these methods do not differentiate between some types of familial and senile systemic amyloidosis.     

Amyloid-like features of polyglutamine aggregates and their assembly kinetics

The repeat length-dependent tendency of the polyglutamine sequences of certain proteins to form aggregates may underlie the cytotoxicity of these sequences in expanded CAG repeat diseases such as Huntington's disease. We report here a number of features of various polyglutamine (polyGln) aggregates and their assembly pathways that bear a resemblance to generally recognized defining features of amyloid fibrils. PolyGln aggregation kinetics displays concentration and length dependence and a lag phase that can be abbreviated by seeding. PolyGln aggregates exhibit classical beta-sheet-rich circular dichroism spectra consistent with an amyloid-like substructure. The fundamental structural unit of all the in vitro aggregates described here is a filament about 3 nm in width, resembling the protofibrillar intermediates in amyloid fibril assembly. We observed these filamentous structures either as isolated threads, as components of ribbonlike sheets, or, rarely, in amyloid-like twisted fibrils. All of the polyGln aggregates described here bind thioflavin T and shift its fluorescence spectrum. Although all polyGln aggregates tested bind the dye Congo red, only aggregates of a relatively long polyGln peptide exhibit Congo red birefringence, and this birefringence is only observed in a small portion of these aggregates. Remarkably, a monoclonal antibody with high selectivity for a generic amyloid fibril conformational epitope is capable of binding polyGln aggregates. Thus, polyGln aggregates exhibit most of the characteristic features of amyloid, but the twisted fibril structure with Congo red birefringence is not the predominant form in the polyGln repeat length range studied here. We also find that polyGln peptides exhibit an unusual freezing-dependent aggregation that appears to be caused by the freeze concentration of peptide and/or buffer components. This is of both fundamental and practical significance. PolyGln aggregation is revealed to be a highly specific process consistent with a significant degree of order in the molecular structure of the product. This ordered structure, or the assembly process leading to it, may be responsible for the cell-specific neuronal degeneration observed in Huntington's and other expanded CAG repeat diseases.     

The binding of thioflavin-T to amyloid fibrils: localisation and implications

Amyloid fibrils are a polymeric form of protein, involving a continuous beta-sheet with the strands perpendicular to the long axis of the fibril. Although typically implicated in diseases such as Alzheimer's disease and the transmissible spongiform encephalopathies, non disease-associated protein can also be converted into amyloid fibrils. Traditionally, amyloid fibrils are identified via the use of specific dyes such as Congo red and thioflavin-T, although their specificity is ill understood. Recently, solutions of bovine insulin and bovine beta-lactoglobulin have been found to form spherulites, micron-sized spherical structures containing radially arranged amyloid fibrils. When studied by confocal microscopy using polarised laser light and thioflavin-T, a consistent pattern of emission, rather than a uniform disc, was observed. This suggests the dye binds in a specific, regular fashion to amyloid fibrils. Confocal microscopy studies of thioflavin-T aligned in stretched poly-vinyl alcohol films showed that the dye dipole excitation axis lies parallel to the long molecular axis. Therefore, thioflavin-T binds to amyloid fibrils such that their long axes are parallel. We propose binding occurs in 'channels' that run along the length of the beta-sheet. Steric interactions between dye molecules and side chains indicate why thioflavin-T fluoresces more intensely when bound to amyloid fibrils and can explain why this interaction with amyloid fibrils is specific, but with varying efficiency.     

The use of supramolecular structures as protein ligands

Congo red dye as well as other eagerly self-assembling organic molecules which form rod-like or ribbon-like supramolecular structures in water solutions, appears to represent a new class of protein ligands with possible wide-ranging medical applications. Such molecules associate with proteins as integral clusters and preferentially penetrate into areas of low molecular stability. Abnormal, partly unfolded proteins are the main binding target for such ligands, while well packed molecules are generally inaccessible. Of particular interest is the observation that local susceptibility for binding supramolecular ligands may be promoted in some proteins as a consequence of function-derived structural changes, and that such complexation may alter the activity profile of target proteins. Examples are presented in this paper.     

Amphoterin includes a sequence motif which is homologous to the Alzheimer's beta-amyloid peptide (Abeta), forms amyloid fibrils in vitro, and binds avidly to Abeta

Many of the proteins associated with amyloidoses have been found to share structural and sequence similarities, which are believed to be responsible for their capability to form amyloid fibrils. Interestingly, some proteins seem to be able to form amyloid-like fibrils although they are not associated with amyloidoses. This indicates that the ability to form amyloid fibrils may be a general property of a greater number of proteins not associated with these diseases. In the present work, we have searched for amyloidogenic consensus sequences in two current protein/peptide databases and show that many proteins share structures which can be predicted to form amyloid. One of these potentially amyloidogenic proteins is amphoterin (also known as HMG-1), involved in neuronal development and a ligand for the receptor for advanced glycation end products (RAGE). It contains an amyloidogenic peptide fragment which is highly homologous to the Alzheimer's amyloid beta-peptide. If enzymatically released from the native protein, it forms amyloid-like fibrils which are visible in electron microscopy, exhibit apple green birefringence under polarized light after Congo red staining, and increases thioflavin T fluorescence. This fragment also shows high affinity to Abeta as a free peptide or while part of the native protein. Our results support the hypothesis that the potential to form amyloid is a common characteristic of a number of proteins, independent of their relation to amyloidoses, and that this potential can be predicted based on the physicochemical properties of these proteins.     

Candida albicans Als adhesins have conserved amyloid-forming sequences

The cell wall-bound Als adhesins of Candida albicans mediate both yeast-to-host tissue adherence and yeast aggregation. This aggregation is amyloid-like, with self-propagating secondary-structure changes, amyloid-characteristic dye binding, and induced birefringence (J. M. Rauceo, N. K. Gaur, K. G. Lee, J. E. Edwards, S. A. Klotz, and P. N. Lipke, Infect. Immun. 72:4948-4955, 2004). Therefore, we determined whether Als proteins could form amyloid fibers with properties like those in cellular aggregation. The beta-aggregation predictor TANGO identified a heptapeptide sequence present in a highly conserved sequence with amyloid-forming potential in Als1p, Als3p, and Als5p. A tridecapeptide containing this sequence formed fibers that bound Congo red and thioflavin T and had characteristic amyloid morphology. Als5p(20-431) and Als5p(20-664), large fragments of Als5p containing the amyloid sequence, also formed amyloid-like fibers and bound Congo red under native conditions. K(a)/K(s) analysis showed that the amyloid-forming sequences are highly conserved in Als proteins and evolve more slowly than other regions of the proteins. Therefore, amyloid-forming ability itself is conserved in these proteins.     

Temperature-dependent, irreversible formation of amyloid fibrils by a soluble human ataxin-3 carrying a moderately expanded polyglutamine stretch (Q36)

The protein ataxin-3 is responsible for Machado-Joseph disease/spinocerebellar ataxia type 3, a neurodegenerative disorder caused by the presence of an expanded polyglutamine tract. A previous investigation [Bevivino, A. E., and Loll, P. J. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 11955-11960] showed that a nonexpanded ataxin-3 (Q27) was fully soluble, whereas an expanded form (Q78) gave rise to amyloid fibrils. Here, we report investigations on three forms of ataxin-3 (i.e., human nonexpanded (Q26), moderately expanded (Q36) ataxins-3, and the murine protein (Q6)). Far-UV circular dichroism spectra at room temperature were substantially similar, with a relatively high helical content. On heating to 96 degrees C, human Q26 and murine proteins did not display large structural changes, nor did they undergo any precipitation, which highlights their amazing heat-resistance. In contrast, human Q36 ataxin-3 underwent a progressive increase in the beta-sheet and a concomitant decrease in helical content when the temperature was shifted from 37 to 80 degrees C, followed by the irreversible formation of aggregates above 80 degrees C. They were shown to consist of amyloid fibrils, as supported by both electron microscopy images and the typical spectral shift displayed by Congo red when it was added to the protein at growing temperatures. We also found that protein precipitation could be prevented by mixing the dye with Q36 ataxin-3 prior to heating, which also confirms that the precipitates do represent authentic amyloid fibrils. In contrast, other compounds structurally related to Congo red did not exert significant effects. Our observations suggest that the temperature of the observed transition is inversely related to the length of the expansion. Finally, we suggest that antiamyloidogenic compounds might be selected on the basis of their ability to block or retard human Q36 ataxin-3 precipitation on heat-treatment.