Anja Böckmann

Structural and functional characterization of two alpha-synuclein strains

α-Synuclein aggregation is implicated in a variety of diseases including Parkinsons disease, dementia with Lewy bodies, pure autonomic failure and multiple system atrophy. The association of protein aggregates made of a single protein with a variety of clinical phenotypes has been explained for prion diseases by the existence of different strains that propagate through the infection pathway. Here we structurally and functionally characterize two polymorphs of α-synuclein. We present evidence that the two forms indeed fulfil the molecular criteria to be identified as two strains of α-synuclein. Specifically, we show that the two strains have different structures, levels of toxicity, and in vitro and in vivo seeding and propagation properties. Such strain differences may account for differences in disease progression in different individuals/cell types and/or types of synucleinopathies.

Atomic-resolution structure of a disease-relevant Aβ(1–42) amyloid fibril

Significance Alzheimer’s disease is the most prevalent neurodegenerative disease still with no known cure. The disease is characterized by the development of extracellular plaques and intracellular neurofibrillary tangles. The senile plaques consist mainly of the peptide amyloid-β (Aβ) in aggregated form, called amyloid fibrils. It is believed that the Aβ amyloid fibrils play an important role in disease progression and cell-to-cell transmissibility, and small Aβ oligomers are often assumed to be the most neurotoxic species. Here, we determined the 3D structure of a disease-relevant Aβ(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, forming a double-horseshoe–like cross–β-sheet entity with maximally buried hydrophobic side chains. Amyloid-β (Aβ) is present in humans as a 39- to 42-amino acid residue metabolic product of the amyloid precursor protein. Although the two predominant forms, Aβ(1–40) and Aβ(1–42), differ in only two residues, they display different biophysical, biological, and clinical behavior. Aβ(1–42) is the more neurotoxic species, aggregates much faster, and dominates in senile plaque of Alzheimer’s disease (AD) patients. Although small Aβ oligomers are believed to be the neurotoxic species, Aβ 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 Aβ(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–β-sheet entity with maximally buried hydrophobic side chains. Residues 1–14 are partially ordered and in a β-strand conformation, but do not display unambiguous distance restraints to the remainder of the core structure.

Atomic-Resolution Three-Dimensional Structure of HET-s(218-289) Amyloid Fibrils by Solid-State NMR Spectroscopy

We present a strategy to solve the high-resolution structure of amyloid fibrils by solid-state NMR and use it to determine the atomic-resolution structure of the prion domain of the fungal prion HET-s in its amyloid form. On the basis of 134 unambiguous distance restraints, we recently showed that HET-s(218-289) in its fibrillar state forms a left-handed β-solenoid, and an atomic-resolution NMR structure of the triangular core was determined from unambiguous restraints only. In this paper, we go considerably further and present a comprehensive protocol using six differently labeled samples, a collection of optimized solid-state NMR experiments, and adapted structure calculation protocols. The high-resolution structure obtained includes the less ordered but biologically important C-terminal part and improves the overall accuracy by including a large number of ambiguous distance restraints.

De Novo 3D Structure Determination from Sub‐milligram Protein Samples by Solid‐State 100 kHz MAS NMR Spectroscopy

Solid-state NMR spectroscopy is an emerging tool for structural studies of crystalline, membrane-associated, sedimented, and fibrillar proteins. A major limitation for many studies is still the large amount of sample needed for the experiments, typically several isotopically labeled samples of 10-20 mg each. Here we show that a new NMR probe, pushing magic-angle sample rotation to frequencies around 100 kHz, makes it possible to narrow the proton resonance lines sufficiently to provide the necessary sensitivity and spectral resolution for efficient and sensitive proton detection. Using restraints from such spectra, a well-defined de novo structure of the model protein ubiquitin was obtained from two samples of roughly 500 μg protein each. This proof of principle opens new avenues for structural studies of proteins available in microgram, or tens of nanomoles, quantities that are, for example, typically achieved for eukaryotic membrane proteins by in-cell or cell-free expression.

Characterization of different water pools in solid-state NMR protein samples

We observed and characterized two distinct signals originating from different pools of water protons in solid-state NMR protein samples, namely from crystal water which exchanges polarization with the protein (on the NMR timescale) and is located in the protein-rich fraction at the periphery of the magic-angle spinning (MAS) sample container, and supernatant water located close to the axis of the sample container. The polarization transfer between the water and the protein can be probed by two-dimensional exchange spectroscopy, and we show that the supernatant water does not interact with protein on the timescale of the experiments. The two water pools have different spectroscopic properties, including resonance frequency, longitudinal, transverse and rotating frame relaxation times. The supernatant water can be removed almost completely physically or can be frozen selectively. Both measures lead to an enhancement of the quality factor of the probe circuit, accompanied by an improvement of the experimental signal/noise, and greatly simplify solvent-suppression by substantially reducing the water signal. We also present a tool, which allows filling solid-state NMR sample containers in a more efficient manner, greatly reducing the amount of supernatant water and maximizing signal/noise.

Proton assisted recoupling and protein structure determination.

We introduce a homonuclear version of third spin assisted recoupling, a second-order mechanism that can be used for polarization transfer between (13)C or (15)N spins in magic angle spinning (MAS) NMR experiments, particularly at high spinning frequencies employed in contemporary high field MAS experiments. The resulting sequence, which we refer to as proton assisted recoupling (PAR), relies on a cross-term between (1)H-(13)C (or (1)H-(15)N) couplings to mediate zero quantum (13)C-(13)C (or (15)N-(15)N recoupling). In particular, using average Hamiltonian theory we derive an effective Hamiltonian for PAR and show that the transfer is mediated by trilinear terms of the form C(1) (+/-)C(2) (-/+)H(Z) for (13)C-(13)C recoupling experiments (or N(1) (+/-)N(2) (-/+)H(Z) for (15)N-(15)N). We use analytical and numerical simulations to explain the structure of the PAR optimization maps and to delineate the PAR matching conditions. We also detail the PAR polarization transfer dependence with respect to the local molecular geometry and explain the observed reduction in dipolar truncation. Finally, we demonstrate the utility of PAR in structural studies of proteins with (13)C-(13)C spectra of uniformly (13)C, (15)N labeled microcrystalline Crh, a 85 amino acid model protein that forms a domain swapped dimer (MW=2 x 10.4 kDa). The spectra, which were acquired at high MAS frequencies (omega(r)2pi>20 kHz) and magnetic fields (750-900 MHz (1)H frequencies) using moderate rf fields, exhibit numerous cross peaks corresponding to long (up to 6-7 A) (13)C-(13)C distances which are particularly useful in protein structure determination. Using results from PAR spectra we calculate the structure of the Crh protein.

A Proton-Detected 4D Solid-State NMR Experiment for Protein Structure Determination

Owing to recent advances in instrumentation, methodology and sample-preparation techniques, solid-state NMR spectroscopy is providing unique insights into biological structures at atomic resolution. Three-dimensional structures of proteins and other biological macromolecules can now be determined that are difficult to characterize by X-ray diffraction or solution NMR. Still, the task remains demanding and new methodological developments are needed to make the structure determination more reliable. While it has been demonstrated that assignments are feasible for proteins with over 200 residues, the structure-determination step remains difficult, mainly because spectral overlap introduces ambiguities in restraint assignments during the process of structure generation. These ambiguities can in some cases be resolved, but remain a potential source of errors. Herein, we present an experimental approach that combines the efficient measurement of longrange proton–proton distances in sparsely isotope-labelled samples with proton-detected 3D and 4D correlation spectroscopy. We demonstrate the method in the context of structure determination of the 76-amino-acid protein ubiquitin. Sparsely distributed protons in an otherwise perdeuterated protein yield highly resolved H spectra. The corresponding samples are produced either by expressing perdeuterated protein followed by H back exchange at about 30% of the exchangeable sites or by using suitable precursors to label exclusively a methyl group of alanine, isoleucine, valine or leucine during protein expression. Under fast magic-angle spinning (55 kHz), coherences in such spin systems are sufficiently long-lived to allow not only dipolar, but also scalar-coupling based polarization transfer. H detection increases the sensitivity by a factor of 8 compared to C detection. Similar deuteration schemes have been exploited in solution-state NMR to increase resolution and sensitivity, and to collect precise NOE restraints. An additional advantage arises in the solid phase. In such samples, even the closest H neighbour often corresponds to a long-range contact and dipolar truncation is not an issue, allowing highly-efficient first-order dipolar-recoupling experiments to be used to obtain distance information. Here we use the dipolar recoupling enhanced by amplitude modulation (DREAM) mixing scheme (Figure 1) that provides particularly high polarization-transfer efficiencies due to its adiabatic nature.

Atomic-Resolution Three-Dimensional Structure of Amyloid β Fibrils Bearing the Osaka Mutation†

Despite its central importance for understanding the molecular basis of Alzheimers 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.

NMR structure and ion channel activity of the p7 protein from hepatitis C virus

The small membrane protein p7 of hepatitis C virus forms oligomers and exhibits ion channel activity essential for virus infectivity. These viroporin features render p7 an attractive target for antiviral drug development. In this study, p7 from strain HCV-J (genotype 1b) was chemically synthesized and purified for ion channel activity measurements and structure analyses. p7 forms cation-selective ion channels in planar lipid bilayers and at the single-channel level by the patch clamp technique. Ion channel activity was shown to be inhibited by hexamethylene amiloride but not by amantadine. Circular dichroism analyses revealed that the structure of p7 is mainly α-helical, irrespective of the membrane mimetic medium (e.g. lysolipids, detergents, or organic solvent/water mixtures). The secondary structure elements of the monomeric form of p7 were determined by 1H and 13C NMR in trifluoroethanol/water mixtures. Molecular dynamics simulations in a model membrane were combined synergistically with structural data obtained from NMR experiments. This approach allowed us to determine the secondary structure elements of p7, which significantly differ from predictions, and to propose a three-dimensional model of the monomeric form of p7 associated with the phospholipid bilayer. These studies revealed the presence of a turn connecting an unexpected N-terminal α-helix to the first transmembrane helix, TM1, and a long cytosolic loop bearing the dibasic motif and connecting TM1 to TM2. These results provide the first detailed experimental structural framework for a better understanding of p7 processing, oligomerization, and ion channel gating mechanism.

The Amyloid–Congo Red Interface at Atomic Resolution

Amyloids are b-sheet-rich proteinaceous aggregates that are a pathological hallmark of a number of human diseases. They can replicate by seeded nucleation and propagate as prions, and may even exert important physiological activities. Amyloids are universally defined by their stainability with Congo red and the resulting green birefringence. Yet, remarkably, the binding mechanism, geometry, and fine structure of the Congo red/amyloid complex are not known. By using solid-state NMR spectroscopy we have characterized, at atomic resolution, the binding interface between Congo red and amyloid fibrils formed by the prion domain of the fungal HET-s protein. The dye binds highly sitespecifically by interacting with residues flanking a groove in the vicinity of a b-arc. The three-dimensional (3D) structure of the fibril is strongly conserved upon the binding of Congo red. Remarkably, a single point mutation, designed according to the binding information, provides an artificial amyloid structurally indistinguishable from HET-s but not stainable by Congo red. The methods used require no isotope labeling of the small molecule and can be used to characterize the interaction of a broad range of dyes, drugs, and tracers with amyloids or other insoluble proteins. Congo red (Figure S1 in the Supporting Information) is a small molecule that reacts specifically with amyloids and has been used since the 1920s as the analytical “gold standard” for amyloid characterization and diagnostics. Despite its binding specificity and its antiamyloidogenic properties, the use of Congo red as a molecular amyloid tracer in vivo and as an antiamyloid drug has been hindered by its high toxicity and poor pharmacokinetics. Contradictory reports exist on the nature of the interaction between Congo red and amyloid fibrils, but the following facts are generally agreed upon: Congo red binds roughly stochiometrically, 9] and the bound form displays a characteristic red-shift in its absorbance maximum, and shows dichroism and birefringence. 10] The negatively charged sulfate groups of Congo red are believed to be relevant for binding to two sites separated by a distance of approximately 20 . In the absence of atomic-resolution structure information, the binding mode is still debated. The long axis of the dye has been postulated to orient parallel or perpendicular to the fibril axis, and the dye binds as a monomer, oligomer, or micelle, or intercalated between the b-sheets. Hydrogen bonds and hydrophobic, aromatic, and ionic interactions have been assumed to contribute to the binding of Congo red to amyloid fibrils. Also, different side chains from residues such as histidine, arginine, and lysine have been suspected to promote congophilia. Atomic-resolution structures of amyloids, which have become available recently, enable studies of the interplay between these proteins and their binding partners in detail. HET-s(218–289), the prion-forming domain of the HET-s prion from the filamentous fungus Podospora anserina, is presently one of the structurally most precisely defined amyloids. To identify the protein surface that interacts with binding partners by NMR spectroscopy, we set out to detect characteristic chemical-shift perturbations (CSP) upon binding by comparing two-dimensional (2D) C–C correlation spectra (proton-driven spin diffusion, PDSD) of stained and unstained [C,N]-labeled fibrils. Since CSPs can be allosteric, we also performed polarization-transfer (PT) experiments which directly probe the spatial proximity of ligand and protein by exploiting the pronounced distance dependence (r ) of the dipolar interaction. Previously published PT approches have proven difficult for our system as they require isotope-labeled Congo red. We have therefore devised a method relying on polarization transfer from ligand H atoms to protein C atoms, which uses standard [H,C,N]-isotope-labeled protein but does not require isotopically labeled ligand. In such samples, the protons from Congo red are the unique polarization source in H–C polarization-transfer experiments, and the C signals detected identify residues in spatial proximity to Congo red (< 4 ). For enhanced spectral resolution, the C atoms are detected in a two-dimensional correlation experiment (dipolar recoupling enhanced by amplitude modulation, DREAM). The main results of the NMR experiments are summarized in Figure 1; details are given in Figures S2–S4 in the [*] A. K. Sch tz, A. Soragni, Dr. M. Ernst, Prof. B. H. Meier Physikalische Chemie, ETH Z rich, 8093 Z rich (Switzerland) E-mail: beme@ethz.ch