Cell-free synthesis of amyloid fibrils with infectious properties and amenable to sub-milligram magic-angle spinning NMR analysis.

Structural investigations of amyloid fibrils often rely on heterologous bacterial overexpression of the protein of interest. Due to their inherent hydrophobicity and tendency to aggregate as inclusion bodies, many amyloid proteins are challenging to express in bacterial systems. Cell-free protein expression is a promising alternative to classical bacterial expression to produce hydrophobic proteins and introduce NMR-active isotopes that can improve and speed up the NMR analysis. Here we implement the cell-free synthesis of the functional amyloid prion HET-s(218-289). We present an interesting case where HET-s(218-289) directly assembles into infectious fibril in the cell-free expression mixture without the requirement of denaturation procedures and purification. By introducing tailored 13C and 15N isotopes or CF3 and 13CH2F labels at strategic amino-acid positions, we demonstrate that cell-free synthesized amyloid fibrils are readily amenable to high-resolution magic-angle spinning NMR at sub-milligram quantity.

Structural dissection of amyloid aggregates of TDP-43 and its C-terminal fragments TDP-35 and TDP-16.

The TAR DNA-binding protein (TDP-43) self-assembles into prion-like aggregates considered to be the structural hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. Here, we use a combination of electron microscopy, X-ray fiber diffraction, Fourier-transform infrared spectroscopy analysis, and solid-state NMR spectroscopy to investigate the molecular organization of different TDP constructs, namely the full-length TDP-43 (1-414), two C-terminal fragments [TDP-35 (90-414) and TDP-16 (267-414)], and a C-terminal truncated fragment (TDP-43 ∆GaroS2), in their fibrillar state. Although the different protein constructs exhibit similar fibril morphology and a typical cross-ß signature by X-ray diffraction, solid-state NMR indicates that TDP-43 and TDP-35 share the same polymorphic molecular structure, while TDP-16 encompasses a well-ordered amyloid core. We identified several residues in the so-called C-terminal GaroS2 (368-414) domain that participates in the rigid core of TDP-16 fibrils, underlining its importance during the aggregation process. Our findings demonstrate that C-terminal fragments can adopt a different molecular conformation in isolation or in the context of the full-length assembly, suggesting that the N-terminal domain and RRM domains play an important role in the TDP-43 amyloid transition.

Molecular architecture of bacterial amyloids in Bacillus biofilms.

The formation of biofilms provides structural and adaptive bacterial response to the environment. In Bacillus species, the biofilm extracellular matrix is composed of exopolysaccharides, hydrophobins, and several functional amyloid proteins. We report, using multiscale approaches such as solid-state NMR (SSNMR), electron microscopy, X-ray diffraction, dynamic light scattering, attenuated total reflection Fourier transform infrared (FTIR), and immune-gold labeling, the molecular architecture of B. subtilis and pathogenic B. cereus functional amyloids. SSNMR data reveal that the major amyloid component TasA in its fibrillar amyloid form contain ß-sheet and α-helical secondary structure, suggesting a nontypical amyloid architecture in B. subtilis. Proteinase K digestion experiments indicate the amyloid moiety is ∼100 aa long, and subsequent SSNMR and FTIR signatures for B. subtilis and B. cereus TasA filaments highlight a conserved amyloid fold, albeit with substantial differences in structural polymorphism and secondary structure composition. Structural analysis and coassembly data on the accessory protein TapA in B. subtilis and its counterpart camelysin in B. cereus reveal a catalyzing effect between the functional amyloid proteins and a common structural architecture, suggesting a coassembly in the context of biofilm formation. Our findings highlight nontypical amyloid behavior of these bacterial functional amyloids, underlining structural variations between biofilms even in closely related bacterial species.-El Mammeri, N., Hierrezuelo, J., Tolchard, J., Cámara-Almirón, J., Caro-Astorga, J., Álvarez-Mena, A., Dutour, A., Berbon, M., Shenoy, J., Morvan, E., Grélard, A., Kauffmann, B., Lecomte, S., de Vicente, A., Habenstein, B., Romero, D., Loquet, A. Molecular architecture of bacterial amyloids in Bacillus biofilms.