Infrared nanospectroscopy characterization of oligomeric and fibrillar aggregates during amyloid formation.
ABSTRACT: Amyloids are insoluble protein fibrillar aggregates. The importance of characterizing their aggregation has steadily increased because of their link to human diseases and material science applications. In particular, misfolding and aggregation of the Josephin domain of ataxin-3 is implicated in spinocerebellar ataxia-3. Infrared nanospectroscopy, simultaneously exploiting atomic force microscopy and infrared spectroscopy, can characterize at the nanoscale the conformational rearrangements of proteins during their aggregation. Here we demonstrate that we can individually characterize the oligomeric and fibrillar species formed along the amyloid aggregation. We describe their secondary structure, monitoring at the nanoscale an ?-to-? transition, and couple these studies with an independent measurement of the evolution of their intrinsic stiffness. These results suggest that the aggregation of Josephin proceeds from the monomer state to the formation of spheroidal intermediates with a native structure. Only successively, these intermediates evolve into misfolded aggregates and into the final fibrils.
Project description:Significant efforts have been devoted in the last twenty years to developing compounds that can interfere with the aggregation pathways of proteins related to misfolding disorders, including Alzheimer's and Parkinson's diseases. However, no disease-modifying drug has become available for clinical use to date for these conditions. One of the main reasons for this failure is the incomplete knowledge of the molecular mechanisms underlying the process by which small molecules interact with protein aggregates and interfere with their aggregation pathways. Here, we leverage the single molecule morphological and chemical sensitivity of infrared nanospectroscopy to provide the first direct measurement of the structure and interaction between single A?42 oligomeric and fibrillar species and an aggregation inhibitor, bexarotene, which is able to prevent A?42 aggregation in vitro and reverses its neurotoxicity in cell and animal models of Alzheimer's disease. Our results demonstrate that the carboxyl group of this compound interacts with A?42 aggregates through a single hydrogen bond. These results establish infrared nanospectroscopy as a powerful tool in structure-based drug discovery for protein misfolding diseases.
Project description:Mid-infrared spectroscopy is a widely used tool for material identification and secondary structure analysis in chemistry, biology and biochemistry. However, the diffraction limit prevents nanoscale protein studies. Here we introduce mapping of protein structure with 30 nm lateral resolution and sensitivity to individual protein complexes by Fourier transform infrared nanospectroscopy (nano-FTIR). We present local broadband spectra of one virus, ferritin complexes, purple membranes and insulin aggregates, which can be interpreted in terms of their ?-helical and/or ?-sheet structure. Applying nano-FTIR for studying insulin fibrils--a model system widely used in neurodegenerative disease research--we find clear evidence that 3-nm-thin amyloid-like fibrils contain a large amount of ?-helical structure. This reveals the surprisingly high level of protein organization in the fibril's periphery, which might explain why fibrils associate. We envision a wide application potential of nano-FTIR, including cellular receptor in vitro mapping and analysis of proteins within quaternary structures.
Project description:Fibrillar aggregation of the protein ataxin-3 is linked to the inherited neurodegenerative disorder Spinocerebellar ataxia type 3, a member of the polyQ expansion disease family. We previously reported that aggregation and stability of the nonpathological form of ataxin-3, carrying an unexpanded polyQ tract, are modulated by its N-terminal Josephin domain. It was also shown that expanded ataxin-3 aggregates via a two-stage mechanism initially involving Josephin self-association, followed by a polyQ-dependent step. Despite this recent progress, however, the exact mechanism of ataxin-3 fibrilization remains elusive. Here, we have used electron microscopy, atomic force microscopy, and other biophysical techniques to characterize the morphological and mechanical properties of nonexpanded ataxin-3 fibrils. By comparing aggregates of ataxin-3 and of the isolated Josephin domain, we show that the two proteins self-assemble into fibrils with markedly similar features over the temperature range 37-50°C. Estimates of persistence length and Young's modulus of the fibrils reveal a great flexibility. Our data indicate that, under physiological conditions, during early aggregation Josephin retains a nativelike secondary structure but loses its enzymatic activity. The results suggest a key role of Josephin in ataxin-3 fibrillar aggregation.
Project description:The morphological features of ?-synuclein (AS) amyloid aggregation in vitro and in cells were elucidated at the nanoscale by far-field subdiffraction fluorescence localization microscopy. Labeling AS with rhodamine spiroamide probes allowed us to image AS fibrillar structures by fluorescence stochastic nanoscopy with an enhanced resolution at least 10-fold higher than that achieved with conventional, diffraction-limited techniques. The implementation of dual-color detection, combined with atomic force microscopy, revealed the propagation of individual fibrils in vitro. In cells, labeled protein appeared as amyloid aggregates of spheroidal morphology and subdiffraction sizes compatible with in vitro supramolecular intermediates perceived independently by atomic force microscopy and cryo-electron tomography. We estimated the number of monomeric protein units present in these minute structures. This approach is ideally suited for the investigation of the molecular mechanisms of amyloid formation both in vitro and in the cellular milieu.
Project description:Infrared nanospectroscopy enables novel possibilities for chemical and structural analysis of nanocomposites, biomaterials or optoelectronic devices. Here we introduce hyperspectral infrared nanoimaging based on Fourier transform infrared nanospectroscopy with a tunable bandwidth-limited laser continuum. We describe the technical implementations and present hyperspectral infrared near-field images of about 5,000 pixel, each one covering the spectral range from 1,000 to 1,900?cm-1. To verify the technique and to demonstrate its application potential, we imaged a three-component polymer blend and a melanin granule in a human hair cross-section, and demonstrate that multivariate data analysis can be applied for extracting spatially resolved chemical information. Particularly, we demonstrate that distribution and chemical interaction between the polymer components can be mapped with a spatial resolution of about 30?nm. We foresee wide application potential of hyperspectral infrared nanoimaging for valuable chemical materials characterization and quality control in various fields ranging from materials sciences to biomedicine.
Project description:Small heat-shock proteins (sHsps) are molecular chaperones that play an important protective role against cellular protein misfolding by interacting with partially unfolded proteins on their off-folding pathway, preventing their aggregation. Polyglutamine (polyQ) repeat expansion leads to the formation of fibrillar protein aggregates and neuronal cell death in nine diseases, including Huntington disease and the spinocerebellar ataxias (SCAs). There is evidence that sHsps have a role in suppression of polyQ-induced neurodegeneration; for example, the sHsp alphaB-crystallin (alphaB-c) has been identified as a suppressor of SCA3 toxicity in a Drosophila model. However, the molecular mechanism for this suppression is unknown. In this study we tested the ability of alphaB-c to suppress the aggregation of a polyQ protein. We found that alphaB-c does not inhibit the formation of SDS-insoluble polyQ fibrils. We further tested the effect of alphaB-c on the aggregation of ataxin-3, a polyQ protein that aggregates via a two-stage aggregation mechanism. The first stage involves association of the N-terminal Josephin domain followed by polyQ-mediated interactions and the formation of SDS-resistant mature fibrils. Our data show that alphaB-c potently inhibits the first stage of ataxin-3 aggregation; however, the second polyQ-dependent stage can still proceed. By using NMR spectroscopy, we have determined that alphaB-c interacts with an extensive region on the surface of the Josephin domain. These data provide an example of a domain/region flanking an amyloidogenic sequence that has a critical role in modulating aggregation of a polypeptide and plays a role in the interaction with molecular chaperones to prevent this aggregation.
Project description:Nanomechanical properties of amyloid fibrils and nanocrystals depend on their secondary and quaternary structure, and the geometry of intermolecular hydrogen bonds. Advanced imaging methods based on atomic force microscopy (AFM) have unravelled the morphological and mechanical heterogeneity of amyloids, however a full understanding has been hampered by the limited resolution of conventional spectroscopic methods. Here, it is shown that single molecule nanomechanical mapping and infrared nanospectroscopy (AFM-IR) in combination with atomistic modelling enable unravelling at the single aggregate scale of the morphological, nanomechanical, chemical, and structural transition from amyloid fibrils to amyloid microcrystals in the hexapeptides, ILQINS, IFQINS, and TFQINS. Different morphologies have different Young's moduli, within 2-6 GPa, with amyloid fibrils exhibiting lower Young's moduli compared to amyloid microcrystals. The origins of this stiffening are unravelled and related to the increased content of intermolecular <i>?</i>-sheet and the increased lengthscale of cooperativity following the transition from twisted fibril to flat nanocrystal. Increased stiffness in Young's moduli is correlated with increased density of intermolecular hydrogen bonding and parallel <i>?</i>-sheet structure, which energetically stabilize crystals over the other polymorphs. These results offer additional evidence for the position of amyloid crystals in the minimum of the protein folding and aggregation landscape.
Project description:Monolayer molybdenum disulfide (MoS2) has received intense interest as a strong candidate for next-generation electronics. However, the observed electrical properties of monolayer MoS2 exhibit several anomalies: samples universally exhibit unexpectedly low mobilities, n-type characteristics, and large contact resistances regardless of contact metal work function. These anomalies have been attributed to the presence of defects, but the mechanism behind this link has been elusive. Here we report the ionization dynamics of sulfur monovacancy defects in monolayer MoS2 probed via noise nanospectroscopy, realized by combining noise-current analysis with atomic force microscopy. Due to the nanoscale dimension of the in situ channel defined by the tip size, we probe a few monovacancy defects at a time. Monovacancy defects exhibit switching between three distinct ionization configurations, corresponding to charge states 0, -1, and -2. The most probable charge configurations are 0 and -1, providing a plausible mechanism to explain the observed anomalies of MoS2 monolayers.
Project description:The chemical and structural properties of biomolecules determine their interactions, and thus their functions, in a wide variety of biochemical processes. Innovative imaging methods have been developed to characterise biomolecular structures down to the angstrom level. However, acquiring vibrational absorption spectra at the single molecule level, a benchmark for bulk sample characterization, has remained elusive. Here, we introduce off-resonance, low power and short pulse infrared nanospectroscopy (ORS-nanoIR) to allow the acquisition of infrared absorption spectra and chemical maps at the single molecule level, at high throughput on a second timescale and with a high signal-to-noise ratio (~10-20). This high sensitivity enables the accurate determination of the secondary structure of single protein molecules with over a million-fold lower mass than conventional bulk vibrational spectroscopy. These results pave the way to probe directly the chemical and structural properties of individual biomolecules, as well as their interactions, in a broad range of chemical and biological systems.
Project description:Aggregation and accumulation of the microtubule-associated protein tau are associated with cognitive decline and neuronal degeneration in Alzheimer's disease and other tauopathies. Thus, preventing the transition of tau from a soluble state to insoluble aggregates and/or reversing the toxicity of existing aggregates would represent a reasonable therapeutic strategy for treating these neurodegenerative diseases. Here we demonstrate that molecular chaperones of the heat shock protein 70 (Hsp70) family are potent inhibitors of tau aggregation in vitro, preventing the formation of both mature fibrils and oligomeric intermediates. Remarkably, addition of Hsp70 to a mixture of oligomeric and fibrillar tau aggregates prevents the toxic effect of these tau species on fast axonal transport, a critical process for neuronal function. When incubated with preformed tau aggregates, Hsp70 preferentially associated with oligomeric over fibrillar tau, suggesting that prefibrillar oligomeric tau aggregates play a prominent role in tau toxicity. Taken together, our data provide a novel molecular basis for the protective effect of Hsp70 in tauopathies.