An Efficient Method of Expression and Purification of Amyloid-Beta (Aß1-42) Peptide from E. coli.

Amyloid-beta (Aß) aggregation into soluble oligomers and fibril formation are associated with Alzheimer's disease (AD) pathogenesis. Aß1-42 is the major form of the Aß peptide present in neuritic plaques and shown to be neurotoxic both in vivo and in vitro. However, understanding the mechanism of its toxicity, aggregation, and other biochemical properties is limited because of its difficult production (recombinant or synthetic) and irreproducibility issues attributed to batch-to-batch preparation differences. Chemically synthetic Aß1-42 is now well established, but it always introduces up to 5% D-isomers along with its L-isomeric form, and thus it is not fruitful for biochemical/structural studies. Here, we optimized an efficient published method for expression and purification of Aß1-42 upon overexpression in Escherichia coli (E. coli) that provides a satisfactory yield as well as minimizes the variability between batch preparations. With the present protocol, ~7-8 mg/liter of unlabeled peptide and ~3.5-4 mg/liter for 13C,15N-labeled (double-labeled) Aß1-42 were obtained.

The three-dimensional structure of human ß-endorphin amyloid fibrils.

In the pituitary gland, hormones are stored in a functional amyloid state within acidic secretory granules before they are released into the blood. To gain a detailed understanding of the structure-function relationship of amyloids in hormone secretion, the three-dimensional (3D) structure of the amyloid fibril of the human hormone ß-endorphin was determined by solid-state NMR. We find that ß-endorphin fibrils are in a ß-solenoid conformation with a protonated glutamate residue in their fibrillar core. During exocytosis of the hormone amyloid the pH increases from acidic in the secretory granule to neutral level in the blood, thus it is suggested-and supported with mutagenesis data-that the pH change in the cellular milieu acts through the deprotonation of glutamate 8 to release the hormone from the amyloid. For amyloid disassembly in the blood, it is proposed that the pH change acts together with a buffer composition change and hormone dilution. In the pituitary gland, peptide hormones can be stored as amyloid fibrils within acidic secretory granules before release into the blood stream. Here, we use solid-state NMR to determine the 3D structure of the amyloid fiber formed by the human hormone ß-endorphin. We find that ß-endorphin fibrils are in a ß-solenoid conformation that is generally reminiscent of other functional amyloids. In the ß-endorphin amyloid, every layer of the ß-solenoid is composed of a single peptide and protonated Glu8 is located in the fibrillar core. The secretory granule has an acidic pH but, on exocytosis, the ß-endorphin fibril would encounter neutral pH conditions (pH 7.4) in the blood; this pH change would result in deprotonation of Glu8 to release the hormone peptide from the amyloid. Analyses of ß-endorphin variants carrying mutations in Glu8 support the role of the protonation state of this residue in fibril disassembly, among other environmental changes.

Quenched hydrogen-deuterium exchange NMR of a disease-relevant Aß(1-42) amyloid polymorph.

Alzheimer's disease is associated with the aggregation into amyloid fibrils of Aß(1-42) and Aß(1-40) peptides. Interestingly, these fibrils often do not obtain one single structure but rather show different morphologies, so-called polymorphs. Here, we compare quenched hydrogen-deuterium (H/D) exchange of a disease-relevant Aß(1-42) fibril for which the 3D structure has been determined by solid-state NMR with H/D exchange previously determined on another structural polymorph. This comparison reveals secondary structural differences between the two polymorphs suggesting that the two polymorphisms can be classified as segmental polymorphs.

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.

NMR-Based Determination of the 3D Structure of the Ligand-Protein Interaction Site without Protein Resonance Assignment.

Molecular replacement in X-ray crystallography is the prime method for establishing structure-activity relationships of pharmaceutically relevant molecules. Such an approach is not available for NMR. Here, we establish a comparable method, called NMR molecular replacement (NMR(2)). The method requires experimentally measured ligand intramolecular NOEs and ligand-protein intermolecular NOEs as well as a previously known receptor structure or model. Our findings demonstrate that NMR(2) may open a new avenue for the fast and robust determination of the interaction site of ligand-protein complexes at atomic resolution.

Solution NMR studies of recombinant Aß(1-42): from the presence of a micellar entity to residual ß-sheet structure in the soluble species.

Amyloid-ß (Aß) peptide is the major component found in senile plaques of Alzheimer's disease patients. The 42-residue fragment Aß(1-42) is proposed to be one of the most pathogenic species therein. Here, the soluble Aß(1-42) species were analyzed by various liquid-state NMR methods. Transient formation of a micelle species was observed at the onset of the aggregation kinetics. This micelle is dissolved after approximately one day. Subsequent loss of this species and the formation of protofibrils are proposed to be the route of fibril formation. Consequently, the observed micelle species is suggested to be on an off-pathway mechanism. Furthermore, characterization of the NMR-observable soluble species shows that it is a random-coil-like entity with low propensities for four ß-strands. These ß-strands correlate with the ß-strand segments observed in Aß fibrils. This finding indicates that the 3D structure of the fibrils might already be predisposed in the soluble species.

Contribution of specific residues of the ß-solenoid fold to HET-s prion function, amyloid structure and stability.

The [Het-s] prion of the fungus Podospora anserina represents a good model system for studying the structure-function relationship in amyloid proteins because a high resolution solid-state NMR structure of the amyloid prion form of the HET-s prion forming domain (PFD) is available. The HET-s PFD adopts a specific ß-solenoid fold with two rungs of ß-strands delimiting a triangular hydrophobic core. A C-terminal loop folds back onto the rigid core region and forms a more dynamic semi-hydrophobic pocket extending the hydrophobic core. Herein, an alanine scanning mutagenesis of the HET-s PFD was conducted. Different structural elements identified in the prion fold such as the triangular hydrophobic core, the salt bridges, the asparagines ladders and the C-terminal loop were altered and the effect of these mutations on prion function, fibril structure and stability was assayed. Prion activity and structure were found to be very robust; only a few key mutations were able to corrupt structure and function. While some mutations strongly destabilize the fold, many substitutions in fact increase stability of the fold. This increase in structural stability did not influence prion formation propensity in vivo. However, if an Ala replacement did alter the structure of the core or did influence the shape of the denaturation curve, the corresponding variant showed a decreased prion efficacy. It is also the finding that in addition to the structural elements of the rigid core region, the aromatic residues in the C-terminal semi-hydrophobic pocket are critical for prion propagation. Mutations in the latter region either positively or negatively affected prion formation. We thus identify a region that modulates prion formation although it is not part of the rigid cross-ß core, an observation that might be relevant to other amyloid models.

Towards a true protein movie: a perspective on the potential impact of the ensemble-based structure determination using exact NOEs.

Confined by the Boltzmann distribution of the energies of the states, a multitude of structural states are inherent to biomolecules. For a detailed understanding of a protein's function, its entire structural landscape at atomic resolution and insight into the interconversion between all the structural states (i.e. dynamics) are required. Whereas dedicated trickery with NMR relaxation provides aspects of local dynamics, and 3D structure determination by NMR is well established, only recently have several attempts been made to formulate a more comprehensive description of the dynamics and the structural landscape of a protein. Here, a perspective is given on the use of exact NOEs (eNOEs) for the elucidation of structural ensembles of a protein describing the covered conformational space.