As the application of high-resolution atomic force microscopy (AFM) has led us recently to the discovery of a unique pressure-induced circular amyloid, we used the same approach to examine morphological events accompanying insulin aggregation under ambient conditions. This study presents the multistage, hierarchical character of the spontaneous fibrillation of insulin at low pH and at 60 and 70 degrees C, and-due to the marked enhancement of image resolution achieved-brings new clues as to the fibrils' ultrastructure and mechanisms of its assembly. Specifically, focusing on the prefibrillar amorphous aggregates occurring 30 s after elevating temperature to the nucleation-enhancing 60 degrees C, revealed the tendency of the globule-shaped oligomers to queue and assembly into elongated forms. This suggests that the shape of the nuclei itself predetermines-in part-the fibrillar architecture of the amyloid. Among first fibrillar features, short but relatively thick (8-nm) seedlike forms appeared on a very short timescale within the first minute of incubation. It has been shown that such fibrils are likely to act as lateral scaffolds for the growth of amyloid. By using phase-image AFM as a nanometer-resolved probe of visco-elastic surface properties, we were able to show that bundles of early protofilaments associated into parallel fibrils are capable of a cooperative transformation into twisted, highly ordered superhelices of the mature amyloid. Independently from producing evidence for the step-resolved character of the process, intermediate and morphologically heterogeneous forms were trapped and characterized, which yields direct evidence for the multipathway character of the amyloidogenesis of insulin. Apart from the faster kinetics, the increased temperature of 70 degrees C leads to a higher degree of morphological variability: along straight rods, twisted ribbonlike structures, rod bundles, and ropelike structures become prominent in the corresponding AFM data.

Pressure perturbation calorimetry (PPC), differential scanning calorimetry (DSC), and time-resolved Fourier transform infrared spectroscopy (FTIR) have been employed to investigate aggregation of bovine insulin at pH 1.9. The aggregation process exhibits two distinguished phases. In the first phase, an intermediate molten globule-like conformational state is transiently formed, reflected by loose tertiary contacts and a robust H/D-exchange. This is followed by unfolding of the native secondary structure. The unfolding of insulin is fast, endothermic, partly reversible, and accompanied by a volume expansion of approximately 0.2%. The second phase consists of actual aggregation: an exothermic irreversible process revealing typical features of nucleation-controlled kinetics. The volumetric changes associated with the second phase are small. The concentration-dependence of DSC scans does not support a monomer intermediate model. While insulin aggregation under ambient pressure is fast and quantitative, pressure as low as 300 bar is sufficient to prevent the aggregation completely, as high-pressure FTIR spectroscopy revealed. This is explained in terms of the high pressure having an adverse effect on the thermal unfolding of insulin, and therefore preventing occurrence of the aggregation-prone intermediate. A comparison of the aggregation in H(2)O and D(2)O shows that the isotopic substitution has diverse effects on both the phases of aggregation. In heavy water, a more pronounced volume expansion accompanies the unfolding stage, while only the second phase shifts to higher temperature.

The presence of 20% (v/v) ethanol triggers growth of insulin amyloid with distinct infrared spectroscopic features, compared with the fibrils obtained under ambient conditions. Here we report that the two insulin amyloid types behave in the prion strain-like manner regarding seeding specificity and ability of the self-propagating conformational template to overrule unfavorable environmental factors and maintain the initial folding pattern. The type of the original seed has been shown to prevail over cosolvent effects and determines spectral position and width of the amide IЈ infrared band of the heterogeneously seeded amyloid. These findings imply that "strains" may be a common generic trait of amyloids.Keywords: insulin; amyloid; prion strains; cross-seeding; protein aggregation Amyloids, which used to be associated with a limited number of pathological conditions, such as Alzheimer disease or the bovine spongiform encephalopathy (BSE)-related Creutzfeldt-Jakob disease, are now thought to represent a common generic feature of proteins as polymers (Fandrich and Dobson 2002). Although comprehensive mechanisms of protein aggregation and amyloidogenesis remain elusive, the problem draws a lot of attention because of its paramount implications in life sciences (Cohen and Kelly 2003).The perplexing observation that a single amino acid sequence of the prion protein propagating in genetically identical hosts may still evoke several distinct phenotypes (both in terms of clinical symptoms and physicochemical properties) of the prion-the so-called prion strains-was initially received as a challenge to the postulated protein-only etiology of the prion disease. The claim as to the common generic character of amyloids as polymers led us to inquire whether the prion strains themselves stem from an analogous common generic trait of amyloids, including those unassociated with prion diseases. To test this hypothesis, we had to find a suitable amyloidogenic protein genetically unrelated to strain-forming prion precursors.Insulin is genetically distant from proteins usually associated with pathological aggregation in vivo, yet it easily forms amyloid in vitro. The wealth of biochemical and structural data has made it an excellent model for amyloid studies (see Nielsen et al. 2001). The insulin aggregation is an energetically self-sustaining process (Dzwolak et al. 2003), which enables carrying out amyloid-seeding experiments at ambient temperature. Seeding amyloid instead of inducing it through en masse destabilization of the native protein (e.g., by prolonged heating) is a more adequate model of an in vivo process. Furthermore, seeding under non-denaturating conditions effectively prevents spontaneous formation of templates other than the originally added seeds themselves. Morphology and infrared spectra of insulin fibrils have been shown to depend on the presence of cosolvents (Nielsen et al. 2001). In this work, two distinct Reprint requests to: Wojciech Dzwolak, High Pressure Research Center, Polish Academy of Sciences...

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Unlike folding, protein aggregation is a multipathway, kinetically controlled process yielding different conformations of fibrils. The dynamics and determinism/indeterminism boundaries of misfolded conformations remain obscure. Here we show that, upon vortexing, insulin forms two distinct types of fibrils with opposite local chiral preferences, which manifest in the opposite twists of bound dye, thioflavin T. Occurrence of either type of fibrils in a test tube is only stochastically determined. By acting through an autocatalytic, "chiral amplification"-like mechanism, a random conformational fluctuation triggers conversion of the macroscopic amount of insulin into aggregates with uniformly biased chiral moieties, which bind and twist likewise the achiral dye. Although a convection-driven chiral amplification in achiral systems, which results in randomly distributed excesses of optically active forms, is known, observation of such a phenomenon in misfolded protein built of l-amino acids is unprecedented. The two optical variants of insulin fibrils show distinct morphologies and can propagate their chiral biases upon seeding to nonagitated insulin solutions. Our findings point to a new aspect of topological complexity of protein fibrils: a chiral feature of hierarchically assembled polypeptides, which is partly emancipated from the innate left-handedness of amino acids. Because altering chirality of a molecule changes dramatically its biological activity, the finding may have important ramifications in the context of the structural basis of "amyloid strains".

Amyloid fibrils, which are often associated with certain degenerative disorders, reveal a number of intriguing spectral properties. However, the relationship between the structure of fibrils and their optical traits remains poorly understood. Poly(L-glutamic) acid is a model polypeptide shown recently to form amyloid-like fibrils with an atypical infrared amide I' band at 1595 cm(-1), which has been attributed to the presence of bifurcated hydrogen bonds coupling C═O and N-D groups of the main chains to glutamate side chains. Here we show that this unusual amide I' band is observed only for fibrils grown from pure enantiomers of the polypeptide, whereas fibrils precipitating from equimolar mixtures of poly(L-glutamic) and poly(D-glutamic) acids have amide I' bands at 1684 and 1612 cm(-1), which are indicative of a typical intermolecular antiparallel β-sheet. Pure enantiomers of polyglutamic acid form spirally twisted superstructures whose handedness is correlated to the amino acid chirality, while fibrils prepared from the racemate do not form scanning electron microscopy (SEM)-detectable mesoscopically ordered structures. Vibrational circular dichroism (VCD) spectra of β-aggregates prepared from mixtures of all L- or D-polyglutamic acid in varying ratios indicate that the enhancement of VCD intensity correlates with the presence of the twisted superstructures. Our results demonstrate that both IR absorption and enhanced VCD are sensitive to subtle packing defects taking place within the compact structure of amyloid fibrils.

A model cosolvent, ethanol, has profound and diversified effects on the amyloidogenic self-assembly of insulin, yielding spectroscopically and morphologically distinguishable forms of beta-aggregates. The alcohol reduces hydrodynamic radii of insulin molecules, decreases enthalpic costs associated with aggregation-prone intermediate states, and accelerates the aggregation itself. Increasing the concentration of the cosolvent promotes curved, amorphous, and finally donut-shaped forms. According to FT-IR data, inter-beta-strand hydrogen bonding is stronger in fibrils formed in the presence of ethanol. Mechanisms underlying the polymorphism of insulin aggregates were investigated by spectroscopic (CD, FT-IR, and fluorescence anisotropy) and calorimetric (DSC and PPC) methods. The nonmonotonic character of the influence of ethanol on insulin aggregation suggests that both preferential exclusion (predominant at the low concentrations) and direct alcohol-protein interactions are involved. The perturbed hydration of aggregation nuclei appears to be a decisive factor in selection of a dominant mode of beta-strand alignment. It may override unfavorable structural consequences of an alternative strand-to-strand stacking, such as strained hydrogen bonding. A hypothetical mechanism of inducing different amyloid "strains" has been put forward. The cooperative character of fibril assembly creates enormous energy barriers for any interstrain transition, which renders the energy landscape comblike-shaped.

The role of microtubule-associated protein Tau in neurodegeneration has been extensively investigated since the discovery of Tau amyloid aggregates in the brains of patients with Alzheimer's disease (AD). The process of formation of amyloid fibrils is known as amyloidogenesis and attracts much attention as a potential target in the prevention and treatment of neurodegenerative conditions linked to protein aggregation. Cerebral deposition of amyloid aggregates of Tau is observed not only in AD but also in numerous other tauopathies and prion diseases. Amyloidogenesis of intrinsically unstructured monomers of Tau can be triggered by mutations in the Tau gene, post-translational modifications, or interactions with polyanionic molecules and aggregation-prone proteins/peptides. The self-assembly of amyloid fibrils of Tau shares a number of characteristic features with amyloidogenesis of other proteins involved in neurodegenerative diseases. For example, in vitro experiments have demonstrated that the nucleation phase, which is the rate-limiting stage of Tau amyloidogenesis, is shortened in the presence of fragmented preformed Tau fibrils acting as aggregation templates ("seeds"). Accordingly, Tau aggregates released by tauopathy-affected neurons can spread the neurodegenerative process in the brain through a prion-like mechanism, originally described for the pathogenic form of prion protein. Moreover, Tau has been shown to form amyloid strains-structurally diverse self-propagating aggregates of potentially various pathological effects, resembling in this respect prion strains. Here, we review the current literature on Tau aggregation and discuss mechanisms of propagation of Tau amyloid in the light of the prion-like paradigm.