Despite its central importance for understanding the molecular basis of Alzheimer's disease (AD), high-resolution structural information on amyloid β-peptide (Aβ) fibrils, which are intimately linked with AD, is scarce. We report an atomic-resolution fibril structure of the Aβ1-40 peptide with the Osaka mutation (E22Δ), associated with early-onset AD. The structure, which differs substantially from all previously proposed models, is based on a large number of unambiguous intra- and intermolecular solid-state NMR distance restraints.

Quantitative solid-state NMR distance measurements in strongly coupled spin systems are often complicated due to the simultaneous presence of multiple noncommuting spin interactions. In the case of zeroth-order homonuclear dipolar recoupling experiments, the recoupled dipolar interaction between distant spins is attenuated by the presence of stronger couplings to nearby spins, an effect known as dipolar truncation. In this article, we quantitatively investigate the effect of dipolar truncation on the polarization-transfer efficiency of various homonuclear recoupling experiments with analytical theory, numerical simulations, and experiments. In particular, using selectively 13 C-labeled tripeptides, we compare the extent of dipolar truncation in model three-spin systems encountered in protein samples produced with uniform and alternating labeling. Our observations indicate that while the extent of dipolar truncation decreases in the absence of directly bonded nuclei, two-bond dipolar couplings can generate significant dipolar truncation of small, long-range couplings. Therefore, while alternating labeling alleviates the effects of dipolar truncation, and thus facilitates the application of recoupling experiments to large spin systems, it does not represent a complete solution to this outstanding problem.

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Inflammasomes are multiprotein complexes that control the innate immune response by activating caspase-1, thus promoting the secretion of cytokines in response to invading pathogens and endogenous triggers. Assembly of inflammasomes is induced by activation of a receptor protein. Many inflammasome receptors require the adapter protein ASC [apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)], which consists of two domains, the N-terminal pyrin domain (PYD) and the C-terminal CARD. Upon activation, ASC forms large oligomeric filaments, which facilitate procaspase-1 recruitment. Here, we characterize the structure and filament formation of mouse ASC in vitro at atomic resolution. Information from cryo-electron microscopy and solid-state NMR spectroscopy is combined in a single structure calculation to obtain the atomic-resolution structure of the ASC filament. Perturbations of NMR resonances upon filament formation monitor the specific binding interfaces of ASC-PYD association. Importantly, NMR experiments show the rigidity of the PYD forming the core of the filament as well as the high mobility of the CARD relative to this core. The findings are validated by structurebased mutagenesis experiments in cultured macrophages. The 3D structure of the mouse ASC-PYD filament is highly similar to the recently determined human ASC-PYD filament, suggesting evolutionary conservation of ASC-dependent inflammasome mechanisms.inflammation | protein structure | protein filament | ASC speck | innate immune response

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Understanding conformational flexibility is of critical importance for understanding protein function, folding, and interactions with other proteins and ligands. NMR spectroscopy is an important tool for such investigations in solution [1] and increasingly also in the solid state [2] since it allows siteresolved studies of dynamic processes. An experimental characterization of all motional modes of a protein is a great challenge and simplified models are necessary. In NMR studies of dynamics, motional amplitudes are generally expressed in terms of a single order parameter, [3] discarding the details of the motion, such as the motional asymmetry. Herein, we show a significant extension of this description, by detecting asymmetric motion of side chains in a protein in the solid state.Dipolar couplings are particularly powerful probes of local molecular dynamics in the solid state. In the absence of motion, the tensor describing the dipolar interaction between two nuclei is a traceless axially symmetric second-rank tensor. It can be characterized by a single parameter, namely its anisotropy d D,rigid which depends only on the internuclear distance and isotope type of the nuclei involved (for the definition see the Supporting Information). In the presence of "fast" motional processes, in other words, processes with a correlation time shorter than approximately 1/d D,rigid , (typically 10-100 ms), the dipolar coupling tensor becomes partially averaged. In the case of a motional process with threefold (C 3 ) or higher symmetry, for example, an isotropic motion within a cone, the averaged tensor remains axially symmetric and is fully characterized by the effective anisotropy d D which has a reduced value compared to d D,rigid . In this case, the motional amplitude can be expressed by a single order parameter [4] However, in the case of a general fast motion, the characterization solely by S is incomplete because the averaged dipolar tensor is no longer axially symmetric [5] and one additional tensor parameter, the asymmetry h D , is needed for a complete description (for the definition, see the Supporting Information). The asymmetry h D varies between zero (symmetric tensor) and one.[5a]In solution-state NMR spectroscopy, dipolar couplings can be measured as residual couplings (RDCs) in anisotropic media. The evaluation of motional amplitudes from RDCs is challenging because RDCs also depend on the (a priori unknown) degree of molecular alignment and the orientation of a given vector relative to the alignment frame and usually data from different alignment media must be combined.[6] The situation is much simplified in solid-state magic-angle-spinning (MAS) NMR, where overall molecular tumbling is absent, allowing the direct measurement of dipolar couplings that depend only on the interatomic distance and dynamics. For the case of one-bond dipolar couplings (CÀH, NÀH, or CÀN) the rigid-limit dipolar coupling tensor is known from the bond lengths. Thus, measurements of the dipolar coupling tensor provide direct access t...

Alzheimer's disease (AD) is the most common form of dementia. Aggregation of amyloid β (Aβ), a peptide of 39-43 residues length, into insoluble fibrils is considered to initiate the disease. Determination of the molecular structure of Aβ fibrils is technically challenging and is a significant goal in AD research that may lead to design of effective therapeutical inhibitors of Aβ aggregation. Here, we present chemical-shift assignments for fibrils formed by highly pure recombinant Aβ1-40 with the Osaka E22Δ mutation that is found in familial AD. We show that that all regions of the peptide are rigid, including the N-terminal part often believed to be flexible in Aβ wt.

Solid-state NMR offers the chance to extend structural studies to proteins that are otherwise difficult to study at atomic resolution, such as protein fibrils, membrane proteins or poorly diffracting crystals. As two-dimensional spatial correlation NMR spectra of proteins suffer from severe resonance overlap, we analyze in this perspective article the potential of higher-dimensional (3D and 4D) proton-detected experiments, which have an increased number of identifiable and assignable distance restraints for solid-state structural studies. We discuss practical considerations for the NMR measurements and the preparation of suitable protein samples and show results of structure calculations from 4D solid-state NMR spectra.