Versatile Near-Infrared Super-Resolution Imaging of Amyloid Fibrils with the Fluorogenic Probe CRANAD-2

CRANAD-2 is a fluorogenic curcumin derivative used for near-infrared detection and imaging in vivo of amyloid aggregates, which are involved in neurodegenerative diseases. We explore the performance of CRANAD-2 in two super-resolution imaging techniques, namely stimulated emission depletion (STED) and single-molecule localization microscopy (SMLM), with markedly different fluorophore requirements. By conveniently adapting the concentration of CRANAD-2, which transiently binds to amyloid fibrils, we show that it performs well in both techniques, achieving a resolution in the range of 45–55 nm. Correlation of SMLM with atomic force microscopy (AFM) validates the resolution of fine features in the reconstructed super-resolved image. The good performance and versatility of CRANAD-2 provides a powerful tool for near-infrared nanoscopic imaging of amyloids in vitro and in vivo.     

Spectral Properties of Thioflavin T and Its Complexes with Amyloid Fibrils

Comparative analysis of the absorption and fluorescence spectra and fluorescence excitation spectra of thioflavin T (ThT) in various solvents and in the composition of amyloid fibrils has shown that ThT, when excited in the region of the long-wavelength absorption band, fluoresces in the spectral region with a maximum at 478–484 nm. The appearance in aqueous and alcohol solutions of a fluorescence band with a maximum near 440 nm has been attributed to the presence in the composition of the ThT preparations of an impurity with an absorption band in the 340–350-nm range. The literature data showing that in glycerol ThT has a wide fluorescence spectrum with two maxima are due to the artifact connected with the use of a high concentration of the dye. It has been suggested that the cause of the low quantum yield of ThT aqueous and alcohol solutions is the breakage of the system of conjugated bonds due to the reorientation of the benzothiozole and benzaminic rings of ThT in the excited state with respect to one another. The main factor determining the high quantum yield of fluorescence of ThT incorporated in fibrils is the steric restriction of the rotation of the rings about one another under these conditions. The suggestions made have been verified by the quantum-chemical calculation of the ThT molecule geometry in the ground and excited states.     

Supramolecular peptide helix from a novel double turn forming peptide containing a β-amino acid

A single-crystal X-ray diffraction study of the terminally protected tetrapeptide Boc-β-Ala-Aib-Leu-Aib-OMe 1 (Aib: α-aminoisobutyric acid; β-Ala: β-Alanine) reveals that it adopts a new type of double turn structure which self-associates to form a unique supramolecular helix through intermolecular hydrogen bonds. Scanning electron microscopic studies show that peptide 1 exhibits amyloid-like fibrillar morphology in the solid state.     

The RHIM of the Immune Adaptor Protein TRIF Forms Hybrid Amyloids with Other Necroptosis-Associated Proteins

TIR-domain-containing adapter-inducing interferon-β (TRIF) is an innate immune protein that serves as an adaptor for multiple cellular signalling outcomes in the context of infection. TRIF is activated via ligation of Toll-like receptors 3 and 4. One outcome of TRIF-directed signalling is the activation of the programmed cell death pathway necroptosis, which is governed by interactions between proteins that contain a RIP Homotypic Interaction Motif (RHIM). TRIF contains a RHIM sequence and can interact with receptor interacting protein kinases 1 (RIPK1) and 3 (RIPK3) to initiate necroptosis. Here, we demonstrate that the RHIM of TRIF is amyloidogenic and supports the formation of homomeric TRIF-containing fibrils. We show that the core tetrad sequence within the RHIM governs the supramolecular organisation of TRIF amyloid assemblies, although the stable amyloid core of TRIF amyloid fibrils comprises a much larger region than the conserved RHIM only. We provide evidence that RHIMs of TRIF, RIPK1 and RIPK3 interact directly to form heteromeric structures and that these TRIF-containing hetero-assemblies display altered and emergent properties that likely underlie necroptosis signalling in response to Toll-like receptor activation.     

Structural transformation from shish‐kebab crystals to micro‐fibrils through hot stretching process of gel‐spun ultra‐high molecular weight polyethylene fibers with high concentration solution

Structural evolution of gel‐spun ultra‐high molecular weight polyethylene fibers with high concentration solution via hot stretching process was investigated by in situ small‐angle X‐ray scattering, in situ wide‐angle X‐ray diffraction measurements, scanning electron microscopy, and differential scanning calorimetry. With the increase of stretching strain, the long period continuously increases at relative lower stretching temperature, while it first increases and then decreases rapidly at relative higher stretching temperature. The kebab thickness almost keeps constant during the whole hot‐stretching process and the kebab diameter continually decreases for all stretching temperatures. Moreover, the length of shish decreases slightly and the shish quantity increases although there is almost no change in the diameter of shish crystals during the hot stretching process. The degree of crystal orientation at different temperatures is as high as above 0.9 during the whole stretching process. These results indicate that the shish‐kebab crystals in ultra‐high molecular weight polyethylene fibers can transform continuously into the micro‐fibril structure composed mostly of shish crystals through the hot stretching process. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2018 , 56, 225–238     

Stepwise Self-assembly of a Tripeptide from Molecular Dimers to Supramolecular β-sheets in Crystals and Amyloid-like Fibrils in the Solid State

The terminally protected tripeptide Boc–Ala(1)–Leu(2)–Ala(3)–OMe 1 forms antiparallel hydrogen-bonded dimers of two different conformers in the asymmetric unit and the individual dimers then self-associate to form supramolecular β-sheet structures in crystals and amyloid-like fibrils in the solid state.     

Effect of the self-assembly of collagen on crystallisation of calcium carbonate in aqueous solution

In order to investigate how the self-assembly of organic matrix influences crystallisation and growth of inorganic minerals, we selected collagen as the matrix and conducted three experiments of crystallisation of CaCO₃ in different reaction systems: H₂O system, as-assembled collagen fibrils system and self-assembling of collagen system. It is found that (i) the self-assembly process of organic matrix had a remarkable effect on the morphology of inorganic minerals: CaCO₃ crystals formed in the as-assembled collagen fibrils system were global clusters and those formed in the self-assembling of collagen system appeared as interlaced networks and (ii) the organic matrix decided the polymorph of crystals: CaCO₃ crystals were calcite in the H₂O system and appeared vaterite in the collagen system. From this study, we can conclude that the self-assembly of collagen fibrils greatly affect the crystallisation and growth of CaCO₃. Such results are significant in understanding the mechanism of biomineralisation in calcified tissues in general, and useful in the synthesis of biominerals.

(a)?CaCO₃ formed in the as-assembled collagen fibrils system. (b)?CaCO₃ formed in the self-assembling of collagen monomer system.The TEM images of samples obtained in the as-assembled collagen fibrils and self-assembling of collagen monomer system, were observed, respectively. The result shows that crystals CaCO₃ formed in the as-assembled collagen fibrils system were global clusters; crystals CaCO₃ formed in the self-assembling of collagen monomer system appeared interlaced networks.     

Fibril Structure Demonstrates the Role of Iodine Labelling on a Pentapeptide Self-Assembly

Iodination has long been employed as a successful labelling strategy to gain structural insights into proteins and other biomolecules via several techniques, including Small Angle X-ray Scattering, Inductively Coupled Plasma Mass Spectrometer (ICP-MS), and single-crystal crystallography. However, when dealing with smaller biomolecular systems, interactions driven by iodine may significantly alter their self-assembly behaviour. The engineering of amyloidogenic peptides for the development of ordered nanomaterials has greatly benefitted from this possibility. Still, to date, iodination has exclusively been applied to aromatic residues. In this work, an aliphatic bis-iodinated amino acid was synthesized and included into a custom pentapeptide, which showed enhanced fibrillogenic behaviour. Peptide single crystal X-ray structure and powder X-ray diffraction on its dried water solution demonstrated the key role of iodine atoms in promoting intermolecular interactions that drive the peptide self-assembly into amyloid fibrils. These findings enlarge the library of halogenated moieties available for directing and engineering the self-assembly of amyloidogenic peptides.     

Binding Modes of Thioflavin T on the Surface of Amyloid Fibrils Studied by NMR

The mechanism for the interaction of thioflavin T (ThT) with amyloid fibrils at the molecular level is not known. Here, we used ¹H NMR spectroscopy to determine the binding mode of ThT on the surface of fibrils from lysozyme and insulin. Relayed rotating‐frame Overhauser enhancements in ThT were observed, indicating that the orientation of ThT is orthogonal to the fibril surface. Importantly, the assembly state of ThT on both surfaces is different. On the surface of insulin fibrils, ThT is oligomeric, as indicated by rapid ¹H spin‐lattice relaxation rate in the rotating frame (R_1ρ), presumably due to intermolecular dipole–dipole interactions between ThT molecules. In contrast, ThT on the surface of lysozyme fibrils is a monomer, as indicated by slower ¹H R_1ρ. These results shed new light into the mechanism for the enhancement of ThT fluorescence and may lead to more efficient detectors of amyloid assemblies, which have escaped detection by ThT in monomer form.