Structural and molecular basis of cross-seeding barriers in amyloids

Neurodegenerative disorders are frequently associated with beta-sheet-rich amyloid deposits. Amyloid-forming proteins can aggregate under different structural conformations known as strains, which can exhibit a prion-like behavior and distinct pathophenotypes. Precise molecular determinants defining strain specificity and cross-strain interactions (cross-seeding) are currently unknown. The HET-s prion protein from the fungus Podospora anserina represents a model system to study the fundamental properties of prion amyloids. Here, we report the amyloid prion structure of HELLF, a distant homolog of the model prion HET-s. We find that these two amyloids, sharing only 17% sequence identity, have nearly identical beta-solenoid folds but lack cross-seeding ability in vivo, indicating that prion specificity can differ in extremely similar amyloid folds. We engineer the HELLF sequence to explore the limits of the sequence-to-fold conservation and to pinpoint determinants of cross-seeding and prion specificity. We find that amyloid fold conservation occurs even at an exceedingly low level of identity to HET-s (5%). Next, we derive a HELLF-based sequence, termed HEC, able to breach the cross-seeding barrier in vivo between HELLF and HET-s, unveiling determinants controlling cross-seeding at residue level. These findings show that virtually identical amyloid backbone structures might not be sufficient for cross-seeding and that critical side-chain positions could determine the seeding specificity of an amyloid fold. Our work redefines the conceptual boundaries of prion strain and sheds light on key molecular features concerning an important class of pathogenic agents

Microstructure Characterization and Creep Deformation of an Al-10 Wt Pct Ti-2 Wt Pct Cu Nanocomposite

The creep behavior of a cryomilled Al-10Ti-2Cu nanocomposite has been studied at temperatures of 533, 588, and 644 K at initial applied stresses ranging from 55 to 117 MPa. Although the strain rates fall within the 10^-10 to 10^-9 S^-1 regime, we observe no evidence of threshold-type creep behavior in this material. We attribute this to the unique microstructure of the present material combined with the mechanism of dislocation slip in ultrafine grain size materials. In particular, the very fine AIN precipitates present within the microstructure are ineffective as obstacles to dislocations during hightemperature deformation. The coherent nature of these fine particles along with their extremely small size prevents a strong dislocation-particle attraction. The inability of the activation energy for self-diffusion in Al to successfully collapse the present creep data onto a single slope combined with the fact that the true activation energy for creep exceeds the value for lattice self-diffusion are both features found in materials containing second-phase particles, which deform simultaneously with the matrix during high-temperature deformation. In the present case, these particles are likely to be Al3Ti

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the propagation of neurodegenerative disorders. Such assemblies consist in multiple protein subunits organized in a non-covalent way to form large macromolecular objects that can execute a variety of cellular functions or cause detrimental consequences. Atomic insights into the assembly mechanisms and the functioning of those macromolecular assemblies remain often scarce since their inherent insolubility and non-crystallinity often drastically reduces the quality of the data obtained from most techniques used in structural biology, such as X-ray crystallography and solution Nuclear Magnetic Resonance (NMR). We here present magic-angle spinning solid-state NMR spectroscopy (SSNMR) as a powerful method to investigate structures of macromolecular assemblies at atomic resolution. SSNMR can reveal atomic details on the assembled complex without size and solubility limitations. The protocol presented here describes the essential steps from the production of 13C/15N isotope-labeled macromolecular protein assemblies to the acquisition of standard SSNMR spectra and their analysis and interpretation. As an example, we show the pipeline of a SSNMR structural analysis of a filamentous protein assembly