Direct amide 15N to 13C transfers for solid-state assignment experiments in deuterated proteins.

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.

Partially-deuterated samples of HET-s(218-289) fibrils: assignment and deuterium isotope effect.

Fast magic-angle spinning and partial sample deuteration allows direct detection of 1H in solid-state NMR, yielding significant gains in mass sensitivity. In order to further analyze the spectra, 1H detection requires assignment of the 1H resonances. In this work, resonance assignments of backbone HN and Hα are presented for HET-s(218-289) fibrils, based on the existing assignment of Cα, Cß, C', and N resonances. The samples used are partially deuterated for higher spectral resolution, and the shifts in resonance frequencies of Cα and Cß due to the deuterium isotope effect are investigated. It is shown that the deuterium isotope effect can be estimated and used for assigning resonances of deuterated samples in solid-state NMR, based on known resonances of the protonated protein.

Atomic-resolution structure of a disease-relevant Aß(1-42) amyloid fibril.

Amyloid-ß (Aß) is present in humans as a 39- to 42-amino acid residue metabolic product of the amyloid precursor protein. Although the two predominant forms, Aß(1-40) and Aß(1-42), differ in only two residues, they display different biophysical, biological, and clinical behavior. Aß(1-42) is the more neurotoxic species, aggregates much faster, and dominates in senile plaque of Alzheimer's disease (AD) patients. Although small Aß oligomers are believed to be the neurotoxic species, Aß amyloid fibrils are, because of their presence in plaques, a pathological hallmark of AD and appear to play an important role in disease progression through cell-to-cell transmissibility. Here, we solved the 3D structure of a disease-relevant Aß(1-42) fibril polymorph, combining data from solid-state NMR spectroscopy and mass-per-length measurements from EM. The 3D structure is composed of two molecules per fibril layer, with residues 15-42 forming a double-horseshoe-like cross-ß-sheet entity with maximally buried hydrophobic side chains. Residues 1-14 are partially ordered and in a ß-strand conformation, but do not display unambiguous distance restraints to the remainder of the core structure.

Solid-state NMR sequential assignment of an Amyloid-ß(1-42) fibril polymorph.

The formation of fibrils of the amyloid-ß (Aß) peptide is considered to be a key event in the pathology of Alzheimer's disease (AD). The determination of a high-resolution structure of these fibrils is relevant for the understanding of the molecular basis of AD. In this work, we present the sequential resonance assignment of one of the polymorphs of Aß(1-42) fibrils. We show that most of the protein is rigid, while a stretch of 4 residues (11-14) is not visible by solid-state NMR spectroscopy due to dynamics.

Sequence-specific solid-state NMR assignments of the mouse ASC PYRIN domain in its filament form.

The apoptosis-associated speck-like protein (ASC protein) plays a central role in eukaryotic innate immune response. Upon infection, multiple ASC molecules assemble into long filaments, which are fundamental for triggering the cellular defense mechanism by starting an inflammatory cascade with the activation of caspase-1. ASC is composed of two domains, the C-terminal caspase-recruitment domain, which is involved in the recruitment of the caspase, and the N-terminal PYRIN domain (PYD), which is responsible for the formation of the filament. Here we present the (13)C and (15)N chemical shift assignment for filaments formed by the PYD of mouse ASC, a 91-residue protein. The backbone between residues 4 and 84 is assigned without interruption. Also, 86 % of the sidechain resonances for this stretch are assigned. Residues 1-3 and 85-91 show unfavorable dynamics and are not observed. Secondary chemical-shift analysis shows the presence of six α-helices.

Solid-state NMR sequential assignments of the N-terminal domain of HpDnaB helicase.

We present solid-state NMR assignments of the N-terminal domain of the DnaB helicase from Helicobacter pylori (153 residues) in its microcrystalline form. We use a sequential resonance assignment strategy based on three-dimensional NMR experiments. The resonance assignments obtained are compared with automated resonance assignments computed with the ssFLYA algorithm. An analysis of the (13)C secondary chemical shifts determines the position of the secondary structure elements in this α-helical protein.

Structure and assembly of the mouse ASC inflammasome by combined NMR spectroscopy and cryo-electron microscopy.

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 structure-based 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.

Solid-state NMR sequential assignments of the C-terminal oligomerization domain of human C4b-binding protein.

The complement 4 binding protein (C4bp) plays a crucial role in the inhibition of the complement cascade. It has an extraordinary seven-arm octopus-like structure with 7 tentacle-like identical chains, held together at their C-terminal end. The C-terminal domain does oligomerize in isolation, and is necessary and sufficient to oligomerize full-length C4bp. It is predicted to form a seven-helix coiled coil, and its multimerization properties make it a promising vaccine adjuvant, probably by enhancing the structural stability and binding affinity of the presented antigen. Here, we present the solid-state NMR resonance assignment of the human C4bp C-terminal oligomerization Domain, hC4pbOD, and the corresponding secondary chemical shifts.

Automated solid-state NMR resonance assignment of protein microcrystals and amyloids.

Solid-state NMR is an emerging structure determination technique for crystalline and non-crystalline protein assemblies, e.g., amyloids. Resonance assignment constitutes the first and often very time-consuming step to a structure. We present ssFLYA, a generally applicable algorithm for automatic assignment of protein solid-state NMR spectra. Application to microcrystals of ubiquitin and the Ure2 prion C-terminal domain, as well as amyloids of HET-s(218-289) and α-synuclein yielded 88-97 % correctness for the backbone and side-chain assignments that are classified as self-consistent by the algorithm, and 77-90 % correctness if also assignments classified as tentative by the algorithm are included.