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The microtubule‐associated protein tau forms disease‐specific filamentous aggregates in several different neurodegenerative diseases. In order to understand how tau undergoes misfolding into a specific filament type and to control this process for drug development purposes, it is crucial to study in vitro tau aggregation methods and investigate the structures of the obtained filaments at the atomic level. Here, we used the tau fragment dGAE, which aggregates spontaneously, to seed the formation of full‐length tau filaments. The structures of dGAE and full‐length tau filaments were investigated by magic‐angle spinning (MAS) solid‐state NMR, showing that dGAE allows propagation of a chronic traumatic encephalopathy (CTE)‐like fold to the full‐length tau. The obtained filaments efficiently seeded tau aggregation in HEK293T cells. This work demonstrates that in vitro preparation of disease‐specific types of full‐length tau filaments is feasible. [ABSTRACT FROM AUTHOR]

Solid-state NMR spectroscopy is a powerful technique to study insoluble and non-crystalline proteins and protein complexes at atomic resolution. The development of proton (1H) detection at fast magic-angle spinning (MAS) has considerably increased the analytical capabilities of the technique, enabling the acquisition of 1H-detected fingerprint experiments in few hours. Here an approach based on double-quantum (DQ) 13C spectroscopy, detected on 1H, is proposed for fast MAS regime (> 60 kHz) to perform the sequential assignment of insoluble proteins of small size, without any specific deuteration requirement. By combining two three-dimensional 1H detected experiments correlating a 13C DQ dimension respectively to its intra-residue and sequential 15 N-1H pairs, a sequential walk through DQ (Ca + CO) resonance is obtained. The approach takes advantage of fast MAS to achieve an efficient sensitivity and the addition of a DQ dimension provides spectral features useful for the resonance assignment process. [ABSTRACT FROM AUTHOR]

The Hepatitis C virus nonstructural protein 5A (NS5A) is a membrane‐associated protein involved in multiple steps of the viral life cycle. Direct‐acting antivirals (DAAs) targeting NS5A are a cornerstone of antiviral therapy, but the mode‐of‐action of these drugs is poorly understood. This is due to the lack of information on the membrane‐bound NS5A structure. Herein, we present the structural model of an NS5A AH‐linker‐D1 protein reconstituted as proteoliposomes. We use highly sensitive proton‐detected solid‐state NMR methods suitable to study samples generated through synthetic biology approaches. Spectra analyses disclose that both the AH membrane anchor and the linker are highly flexible. Paramagnetic relaxation enhancements (PRE) reveal that the dimer organization in lipids requires a new type of NS5A self‐interaction not reflected in previous crystal structures. In conclusion, we provide the first characterization of NS5A AH‐linker‐D1 in a lipidic environment shedding light onto the mode‐of‐action of clinically used NS5A inhibitors. [ABSTRACT FROM AUTHOR]

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‐β 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. [ABSTRACT FROM AUTHOR]

The assignment of protein backbone and side-chain NMR chemical shifts is the first step towards the characterization of protein structure. The recent introduction of proton detection in combination with fast MAS has opened up novel opportunities for assignment experiments. However, typical 3D sequential-assignment experiments using proton detection under fast MAS lead to signal intensities much smaller than the theoretically expected ones due to the low transfer efficiency of some of the steps. Here, we present a selective 3D experiment for deuterated and (amide) proton back-exchanged proteins where polarization is directly transferred from backbone nitrogen to selected backbone or sidechain carbons. The proposed pulse sequence uses only 1H-15N cross-polarization (CP) transfers, which are, for deuterated proteins, about 30% more efficient than 1H-13C CP transfers, and employs a dipolar version of the INEPT experiment for N-C transfer. By avoiding HN-C (HN stands for amide protons) and C-C CP transfers, we could achieve higher selectivity and increased signal intensities compared to other pulse sequences containing long-range CP transfers. The REDOR transfer is designed with an additional selective π pulse, which enables the selective transfer of the polarization to the desired 13C spins. [ABSTRACT FROM AUTHOR]

15N R1ρ relaxation experiments in solid-state NMR spectroscopy are sensitive to timescales and amplitudes of internal protein motions in the hundreds of nano- to microsecond time window, which is difficult to probe by solution-state NMR spectroscopy. By using 15N R1ρ relaxation experiments, a simplified approach to detect low microsecond protein dynamics is described and residue-specific correlation times are determined from the ratio of 15N R1ρ rate constants at different magic angle spinning frequencies. Microcrystalline ubiquitin exhibits small-amplitude dynamics on a timescale of about 1 μs across the entire protein, and larger amplitude motions, also on the 1 μs timescale, for several sites, including the β1-β2 turn and the N terminus of the α helix. According to the analysis, the microsecond protein backbone dynamics are of lower amplitude than that concluded in previous solid-state NMR spectroscopy studies, but persist across the entire protein with a rather uniform timescale of 1 μs. [ABSTRACT FROM AUTHOR]

ABSTRACT 1-( N, N-substituted-aminiomethyl)spirobi[3-oxo(2,5-dioxa-1-silacyclopentan)]ates were investigated using NMR, X-ray, and quantum-chemical methods. The free activation parameters for the inversion of 'ammonium-amine nitrogen' and the chirality change at the pentacoordinated silicon were determined by dynamic NMR spectroscopy. The latter proceeds via the dissociation of SiO bonds. The results were confirmed by quantum-chemical calculations, the experimental observation of cross-peaks in 2D-EXSY NMR spectra, and the spirocycle exchange reactions. Transition pathways between the different diastereomers in solution were analyzed. [ABSTRACT FROM AUTHOR]

Spider silk is renowned for its exceptional mechanical properties, surpassing those of other natural and many synthetic fibers. Yet, replicating its remarkable properties through synthetic production remains a challenge. The variability in the mechanical properties of synthetic spider silks lacking protective coatings, exacerbated by factors such as spinning conditions and humidity levels, poses an additional challenge, impacting their application potential. Bioconjugation offers a versatile synthetic method to modify protein structures, enhancing their pharmacokinetics, solubility, stability, and immune response. In particular, polyethylene glycol (PEG)‐ylation has emerged as a successful strategy with numerous marketed PEG–protein conjugates. This study introduces synthetic spider silk—multiarm PEG bioconjugates, facilitating spidroin crosslinking, and chemical functionalization while retaining a biomimetic spinning approach. Two different examples demonstrate the potential of this approach to improve the fiber's tensile strength and extensibility, respectively, both leading to an increased toughness modulus. Furthermore, the approach could allow the tuning of fiber mechanical properties without developing a new mini‐spidroin construct and fiber coating with lipids attached to multiarm PEG, potentially mitigating the impact of environmental conditions on synthetic spider silk fibers. [ABSTRACT FROM AUTHOR]