The unique enhanced sensitivity of vibrational circular dichroism (VCD) to the formation and development of amyloid fibrils in solution is extended to four additional fibril-forming proteins or peptides where it is shown that the sign of the fibril VCD pattern correlates with the sense of supramolecular filament chirality and, without exception, to the dominant fibril morphology as observed in AFM or SEM images. Previously for insulin, it has been demonstrated that the sign of the VCD band pattern from filament chirality can be controlled by adjusting the pH of the incubating solution, above pH 2 for “normal” left-hand-helical filaments and below pH 2 for “reversed” right-hand-helical filaments. From AFM or SEM images, left-helical filaments form multifilament braids of left-twisted fibrils while the right-helical filaments form parallel filament rows of fibrils with a flat tape-like morphology, the two major classes of fibril morphology that from deep UV resonance Raman scattering exhibit the same cross-β-core secondary structure. Here we investigate whether fibril supramolecular chirality is the underlying cause of the major morphology differences in all amyloid fibrils by showing that the morphology (twisted versus flat) of fibrils of lysozyme, apo-α-lactalbumin, HET-s (218–289) prion, and a short polypeptide fragment of transthyretin, TTR (105–115), directly correlates to their supramolecular chirality as revealed by VCD. The result is strong evidence that the chiral supramolecular organization of filaments is the principal underlying cause of the morphological heterogeneity of amyloid fibrils. Because fibril morphology is linked to cell toxicity, the chirality of amyloid aggregates should be explored in the widely used in vitro models of amyloid-associated diseases.

The aggresome is an organelle that recruits aggregated proteins for storage and degradation. We performed an siRNA screen for proteins involved in aggresome formation and identified novel mammalian AAA+ protein disaggregases RuvbL1 and RuvbL2. Depletion of RuvbL1 or RuvbL2 suppressed aggresome formation and caused buildup of multiple cytoplasmic aggregates. Similarly, downregulation of RuvbL orthologs in yeast suppressed the formation of an aggresome-like body and enhanced the aggregate toxicity. In contrast, their overproduction enhanced the resistance to proteotoxic stress independently of chaperone Hsp104. Mammalian RuvbL associated with the aggresome, and the aggresome substrate synphilin-1 interacted directly with the RuvbL1 barrel-like structure near the opening of the central channel. Importantly, polypeptides with unfolded structures and amyloid fibrils stimulated the ATPase activity of RuvbL. Finally, disassembly of protein aggregates was promoted by RuvbL. These data indicate that RuvbL complexes serve as chaperones in protein disaggregation.

Chaperonins mediate protein folding in a cavity formed by multisubunit rings. The human CCT has eight non-identical subunits and the His147Arg mutation in one subunit, CCT5, causes neuropathy. Knowledge is scarce on the impact of this and other mutations upon the chaperone's structure and functions. To make progress, experimental models must be developed. We used an archaeal mutant homolog and demonstrated that the His147Arg mutant has impaired oligomeric assembly, ATPase activity, and defective protein homeostasis functions. These results establish for the first time that a human chaperonin gene defect can be reproduced and studied at the molecular level with an archaeal homolog. The major advantage of the system, consisting of rings with eight identical subunits, is that it amplifies the effects of a mutation as compared with the human counterpart, in which just one subunit per ring is defective. Therefore, the slight deficit of a non-lethal mutation can be detected and characterized.

In this combined experimental (deep ultraviolet resonance Raman (DUVRR) spectroscopy and atomic force microscopy (AFM)) and theoretical (molecular dynamics (MD) simulations and stress–strain (SS)) study, the structural and mechanical properties of amyloid beta (Aβ40) fibrils have been investigated. The DUVRR spectroscopy and AFM experiments confirmed the formation of linear, unbranched and β-sheet rich fibrils. The fibrils (Aβ40)n, formed using n monomers, were equilibrated using all-atom MD simulations. The structural properties such as β-sheet character, twist, interstrand distance, and periodicity of these fibrils were found to be in agreement with experimental measurements. Furthermore, Young’s modulus (Y) = 4.2 GPa computed using SS calculations was supported by measured values of 1.79 ± 0.41 and 3.2 ± 0.8 GPa provided by two separate AFM experiments. These results revealed size dependence of structural and material properties of amyloid fibrils and show the utility of such combined experimental and theoretical studies in the design of precisely engineered biomaterials.

MXenes are among the most widely researched materials due to a unique combination of high electronic conductivity and hydrophilic surface, confined in a 2D structure. Therefore, comprehensive characterization of individual MXene flakes is of great importance. Here we report on nanoscale Raman imaging of single-layer and few-layer flakes of Ti3C2T x MXene deposited on a gold substrate using tip-enhanced Raman scattering (TERS). TERS spectra of MXene monolayers are dominated by an intense peak at around 201 cm–1 and two well-defined peaks at around 126 and 725 cm–1. Absolute intensities of these peaks decrease with increasing number of layers, though the relative intensity of the 126 and 725 cm–1 bands as compared to the 201 cm–1 band increases. The peak positions of the main MXene bands do not significantly change in flakes of different number of layers, suggesting weak coupling between the MXene layers. In addition, we observed stiffening of the 201 cm–1 vibration over the wrinkles in MXene flakes. Using TERS for nanoscale spectroscopic characterization of Ti3C2T x allows fast Raman mapping with deep subdiffraction resolution at the laser power density on the sample about an order of magnitude lower as compared to confocal Raman measurements. Finally, we demonstrate very high environmental stability of stoichiometric single-layer MXenes and show that the intensity of TERS response from the single- and few-layer flakes of Ti3C2T x can be used to track early stages of degradation, well before significant morphological changes appear.

Amyloid fibril polymorphism is not well understood despite its potential importance for biological activity and associated toxicity. Controlling the polymorphism of mature fibrils including their morphology and supramolecular chirality by postfibrillation changes in the local environment is the subject of this study. Specifically, the effect of pH on the stability and dynamics of HET-s (218-289) prion fibrils has been determined through the use of vibrational circular dichroism (VCD), deep UV resonance Raman, and fluorescence spectroscopies. It was found that a change in solution pH causes deprotonation of Asp and Glu amino acid residues on the surface of HET-s (218-289) prion fibrils and triggers rapid transformation of one supramolecular chiral polymorph into another. This process involves changes in higher order arrangements like lateral filament and fibril association and their supramolecular chirality, while the fibril cross-β core remains intact. This work suggests a hypothetical mechanism for HET-s (218-289) prion fibril refolding and proposes that the interconversion between fibril polymorphs driven by the solution environment change is a general property of amyloid fibrils.

Supramolecular chirality of amyloid fibrils, protein aggregates related to many neurodegenerative diseases, is a remarkable property associated with fibril structure and polymorphism. Since its discovery almost 10 years ago there is still little understanding of this phenomenon, including the cause of the highly enhanced vibrational circular dichroism (VCD) intensity arising from fibril supramolecular chirality. In this study, VCD spectra, enhanced by filament supramolecular chirality, are presented for lysozyme and insulin fibrils above and below pH 2 and after deuterium exchange, above and below pD 2. Supramolecular chirality (observed by VCD) and fibril morphology (documented by atomic force microscopy) are not affected by protein deuteriation. In D O the fibril VCD sign pattern changes to fewer bands, with implications for the amide I/II origin of enhanced VCD intensity. Separation of amide I and II signals will facilitate calculations of enhanced VCD spectra of amyloid fibrils and enable a better understanding of the origin of the VCD sign pattern.

A purple color is formed during the fibrillation of lysozyme, a well-studied protein lacking a prosthetic group. The application of Raman spectroscopy, electron paramagnetic resonance and UV-vis absorption spectroscopy indicates the formation of a sulfur∴π-bonded radical cation due to the methionine-phenylalanine interaction, which is consistent with a small molecule model reported in the literature. A purple chromophore with characteristic 550 nm absorption is formed due to a specific orientation of the sulfur-centered radical cation and a phenyl ring stabilized by the fibril framework. A specific fibril conformation and the resulting formation of the chromophore are controlled reversibly by varying the pH. This is the first known example of a side chain self-assembled chromophore formed due to protein aggregation.