Robert Tycko

A structural model for Alzheimer's β-amyloid fibrils based on experimental constraints from solid state NMR

We present a structural model for amyloid fibrils formed by the 40-residue β-amyloid peptide associated with Alzheimers disease (Aβ1–40), based on a set of experimental constraints from solid state NMR spectroscopy. The model additionally incorporates the cross-β structural motif established by x-ray fiber diffraction and satisfies constraints on Aβ1–40 fibril dimensions and mass-per-length determined from electron microscopy. Approximately the first 10 residues of Aβ1–40 are structurally disordered in the fibrils. Residues 12–24 and 30–40 adopt β-strand conformations and form parallel β-sheets through intermolecular hydrogen bonding. Residues 25–29 contain a bend of the peptide backbone that brings the two β-sheets in contact through sidechain-sidechain interactions. A single cross-β unit is then a double-layered β-sheet structure with a hydrophobic core and one hydrophobic face. The only charged sidechains in the core are those of D23 and K28, which form salt bridges. Fibrils with minimum mass-per-length and diameter consist of two cross-β units with their hydrophobic faces juxtaposed.

Cell-free Formation of RNA Granules: Low Complexity Sequence Domains Form Dynamic Fibers within Hydrogels

Eukaryotic cells contain assemblies of RNAs and proteins termed RNA granules. Many proteins within these bodies contain KH or RRM RNA-binding domains as well as low complexity (LC) sequences of unknown function. We discovered that exposure of cell or tissue lysates to a biotinylated isoxazole (b-isox) chemical precipitated hundreds of RNA-binding proteins with significant overlap to the constituents of RNA granules. The LC sequences within these proteins are both necessary and sufficient for b-isox-mediated aggregation, and these domains can undergo a concentration-dependent phase transition to a hydrogel-like state in the absence of the chemical. X-ray diffraction and EM studies revealed the hydrogels to be composed of uniformly polymerized amyloid-like fibers. Unlike pathogenic fibers, the LC sequence-based polymers described here are dynamic and accommodate heterotypic polymerization. These observations offer a framework for understanding the function of LC sequences as well as an organizing principle for cellular structures that are not membrane bound.

Molecular structural basis for polymorphism in Alzheimer's β-amyloid fibrils

We describe a full structural model for amyloid fibrils formed by the 40-residue β-amyloid peptide associated with Alzheimers disease (Aβ1–40), based on numerous constraints from solid state NMR and electron microscopy. This model applies specifically to fibrils with a periodically twisted morphology, with twist period equal to 120 ± 20 nm (defined as the distance between apparent minima in fibril width in negatively stained transmission electron microscope images). The structure has threefold symmetry about the fibril growth axis, implied by mass-per-length data and the observation of a single set of 13C NMR signals. Comparison with a previously reported model for Aβ1–40 fibrils with a qualitatively different, striated ribbon morphology reveals the molecular basis for polymorphism. At the molecular level, the 2 Aβ1–40 fibril morphologies differ in overall symmetry (twofold vs. threefold), the conformation of non-β-strand segments, and certain quaternary contacts. Both morphologies contain in-register parallel β-sheets, constructed from nearly the same β-strand segments. Because twisted and striated ribbon morphologies are also observed for amyloid fibrils formed by other polypeptides, such as the amylin peptide associated with type 2 diabetes, these structural variations may have general implications.

Molecular Structure of β-Amyloid Fibrils in Alzheimer’s Disease Brain Tissue

In vitro, β-amyloid (Aβ) peptides form polymorphic fibrils, with molecular structures that depend on growth conditions, plus various oligomeric and protofibrillar aggregates. Here, we investigate structures of human brain-derived Aβ fibrils, using seeded fibril growth from brain extract and data from solid-state nuclear magnetic resonance and electron microscopy. Experiments on tissue from two Alzheimers disease (AD) patients with distinct clinical histories showed a single predominant 40 residue Aβ (Aβ40) fibril structure in each patient; however, the structures were different from one another. A molecular structural model developed for Aβ40 fibrils from one patient reveals features that distinguish in-vivo- from in-vitro-produced fibrils. The data suggest that fibrils in the brain may spread from a single nucleation site, that structural variations may correlate with variations in AD, and that structure-specific amyloid imaging agents may be an important future goal.

Molecular structure of amyloid fibrils: insights from solid-state NMR.

Solid-state nuclear magnetic resonance (NMR) measurements have made major contributions to our understanding of the molecular structures of amyloid fibrils, including fibrils formed by the beta-amyloid peptide associated with Alzheimers disease, by proteins associated with fungal prions, and by a variety of other polypeptides. Because solid-state NMR techniques can be used to determine interatomic distances (both intramolecular and intermolecular), place constraints on backbone and side-chain torsion angles, and identify tertiary and quaternary contacts, full molecular models for amyloid fibrils can be developed from solid-state NMR data, especially when supplemented by lower-resolution structural constraints from electron microscopy and other sources. In addition, solid-state NMR data can be used as experimental tests of various proposals and hypotheses regarding the mechanisms of amyloid formation, the nature of intermediate structures, and the common structural features within amyloid fibrils. This review introduces the basic experimental and conceptual principles behind solid-state NMR methods that are applicable to amyloid fibrils, reviews the information about amyloid structures that has been obtained to date with these methods, and discusses how solid-state NMR data provide insights into the molecular interactions that stabilize amyloid structures, the generic propensity of polypeptide chains to form amyloid fibrils, and a number of related issues that are of current interest in the amyloid field.

Supramolecular Structure in Full-Length Alzheimer's β-Amyloid Fibrils: Evidence for a Parallel β-Sheet Organization from Solid-State Nuclear Magnetic Resonance

Abstract We report constraints on the supramolecular structure of amyloid fibrils formed by the 40-residue β -amyloid peptide associated with Alzheimers disease (A β 1–40 ) obtained from solid-state nuclear magnetic resonance (NMR) measurements of intermolecular dipole-dipole couplings between 13 C labels at 11 carbon sites in residues 2 through 39. The measurements are carried out under magic-angle spinning conditions, using the constant-time finite-pulse radiofrequency-driven recoupling (fpRFDR-CT) technique. We also present one-dimensional 13 C magic-angle spinning NMR spectra of the labeled A β 1–40 samples. The fpRFDR-CT data reveal nearest-neighbor intermolecular distances of 4.8±0.5A for carbon sites from residues 12 through 39, indicating a parallel alignment of neighboring peptide chains in the predominantly β -sheet structure of the amyloid fibrils. The one-dimensional NMR spectra indicate structural order at these sites. The fpRFDR-CT data and NMR spectra also indicate structural disorder in the N-terminal segment of A β 1–40 , including the first nine residues. These results place strong constraints on any molecular-level structural model for full-length β -amyloid fibrils.

Amyloid of the prion domain of Sup35p has an in-register parallel β-sheet structure

The [PSI+] prion of Saccharomyces cerevisiae is a self-propagating amyloid form of Sup35p, a subunit of the translation termination factor. Using solid-state NMR we have examined the structure of amyloid fibrils formed in vitro from purified recombinant Sup351–253, consisting of the glutamine- and asparagine-rich N-terminal 123-residue prion domain (N) and the adjacent 130-residue highly charged M domain. Measurements of magnetic dipole–dipole couplings among 13C nuclei in a series of Sup35NM fibril samples, 13C-labeled at backbone carbonyl sites of Tyr, Leu, or Phe residues or at side-chain methyl sites of Ala residues, indicate intermolecular 13C–13C distances of ≈0.5 nm for nearly all sites in the N domain. Certain sites in the M domain also exhibit intermolecular distances of ≈0.5 nm. These results indicate that an in-register parallel β-sheet structure underlies the [PSI+] prion phenomenon.

Seeded growth of β-amyloid fibrils from Alzheimer's brain-derived fibrils produces a distinct fibril structure

Studies by solid-state nuclear magnetic resonance (NMR) of amyloid fibrils prepared in vitro from synthetic 40-residue β-amyloid (Aβ1–40) peptides have shown that the molecular structure of Aβ1–40 fibrils is not uniquely determined by amino acid sequence. Instead, the fibril structure depends on the precise details of growth conditions. The molecular structures of β-amyloid fibrils that develop in Alzheimers disease (AD) are therefore uncertain. We demonstrate through thioflavin T fluorescence and electron microscopy that fibrils extracted from brain tissue of deceased AD patients can be used to seed the growth of synthetic Aβ1–40 fibrils, allowing preparation of fibrils with isotopic labeling and in sufficient quantities for solid-state NMR and other measurements. Because amyloid structures propagate themselves in seeded growth, as shown in previous studies, the molecular structures of brain-seeded synthetic Aβ1–40 fibrils most likely reflect structures that are present in AD brain. Solid-state 13C NMR spectra of fibril samples seeded with brain material from two AD patients were found to be nearly identical, indicating the same molecular structures. Spectra of an unseeded control sample indicate greater structural heterogeneity. 13C chemical shifts and other NMR data indicate that the predominant molecular structure in brain-seeded fibrils differs from the structures of purely synthetic Aβ1–40 fibrils that have been characterized in detail previously. These results demonstrate a new approach to detailed structural characterization of amyloid fibrils that develop in human tissue, and to investigations of possible correlations between fibril structure and the degree of cognitive impairment and neurodegeneration in AD.

Molecular Structures of Amyloid and Prion Fibrils: Consensus versus Controversy

Many peptides and proteins self-assemble into amyloid fibrils. Examples include mammalian and fungal prion proteins, polypeptides associated with human amyloid diseases, and proteins that may have biologically functional amyloid states. To understand the propensity for polypeptides to form amyloid fibrils and to facilitate rational design of amyloid inhibitors and imaging agents, it is necessary to elucidate the molecular structures of these fibrils. Although fibril structures were largely mysterious 15 years ago, a considerable body of reliable structural information about amyloid fibril structures now exists, with essential contributions from solid state nuclear magnetic resonance (NMR) measurements. This Account reviews results from our laboratories and discusses several structural issues that have been controversial. In many cases, the amino acid sequences of amyloid fibrils do not uniquely determine their molecular structures. Self-propagating, molecular-level polymorphism complicates the structure determination problem and can lead to apparent disagreements between results from different laboratories, particularly when different laboratories study different polymorphs. For 40-residue β-amyloid (Aβ₁₋₄₀) fibrils associated with Alzheimers disease, we have developed detailed structural models from solid state NMR and electron microscopy data for two polymorphs. These polymorphs have similar peptide conformations, identical in-register parallel β-sheet organizations, but different overall symmetry. Other polymorphs have also been partially characterized by solid state NMR and appear to have similar structures. In contrast, cryo-electron microscopy studies that use significantly different fibril growth conditions have identified structures that appear (at low resolution) to be different from those examined by solid state NMR. Based on solid state NMR and electron paramagnetic resonance (EPR) measurements, the in-register parallel β-sheet organization found in β-amyloid fibrils also occurs in many other fibril-forming systems. We attribute this common structural motif to the stabilization of amyloid structures by intermolecular interactions among like amino acids, including hydrophobic interactions and polar zippers. Surprisingly, we have recently identified and characterized antiparallel β-sheets in certain fibrils that are formed by the D23N mutant of Aβ₁₋₄₀, a mutant that is associated with early-onset, familial neurodegenerative disease. Antiparallel D23N-Aβ₁₋₄₀ fibrils are metastable with respect to parallel structures and, therefore, represent an off-pathway intermediate in the amyloid fibril formation process. Other methods have recently produced additional evidence for antiparallel β-sheets in other amyloid-formation intermediates. As an alternative to simple parallel and antiparallel β-sheet structures, researchers have proposed β-helical structural models for some fibrils, especially those formed by mammalian and fungal prion proteins. Solid state NMR and EPR data show that fibrils formed in vitro by recombinant PrP have in-register parallel β-sheet structures. However, the structure of infectious PrP aggregates is not yet known. The fungal HET-s prion protein has been shown to contain a β-helical structure. However, all yeast prions studied by solid state NMR (Sup35p, Ure2p, and Rnq1p) have in-register parallel β-sheet structures, with their Gln- and Asn-rich N-terminal segments forming the fibril core.

Antiparallel β-sheet architecture in Iowa-mutant β-amyloid fibrils

Wild-type, full-length (40- and 42-residue) amyloid β-peptide (Aβ) fibrils have been shown by a variety of magnetic resonance techniques to contain cross-β structures in which the β-sheets have an in-register parallel supramolecular organization. In contrast, recent studies of fibrils formed in vitro by the Asp23-to-Asn mutant of 40-residue Aβ (D23N-Aβ1–40), which is associated with early onset neurodegeneration, indicate that D23N-Aβ1–40 fibrils can contain either parallel or antiparallel β-sheets. We report a protocol for producing structurally pure antiparallel D23N-Aβ1–40 fibril samples and a series of solid state nuclear magnetic resonance and electron microscopy measurements that lead to a specific model for the antiparallel D23N-Aβ1–40 fibril structure. This model reveals how both parallel and antiparallel cross-β structures can be constructed from similar peptide monomer conformations and stabilized by similar sets of interactions, primarily hydrophobic in nature. We find that antiparallel D23N-Aβ1–40 fibrils are thermodynamically metastable with respect to conversion to parallel structures, propagate less efficiently than parallel fibrils in seeded fibril growth, and therefore must nucleate more efficiently than parallel fibrils in order to be observable. Experiments in neuronal cell cultures indicate that both antiparallel and parallel D23N-Aβ1–40 fibrils are cytotoxic. Thus, our antiparallel D23N-Aβ1–40 fibril model represents a specific “toxic intermediate” in the aggregation process of a disease-associated Aβ mutant.