Henrike Heise

Pre-fibrillar α-synuclein variants with impaired β-structure increase neurotoxicity in Parkinson's disease models

The relation of α‐synuclein (αS) aggregation to Parkinsons disease (PD) has long been recognized, but the mechanism of toxicity, the pathogenic species and its molecular properties are yet to be identified. To obtain insight into the function different aggregated αS species have in neurotoxicity in vivo, we generated αS variants by a structure‐based rational design. Biophysical analysis revealed that the αS mutants have a reduced fibrillization propensity, but form increased amounts of soluble oligomers. To assess their biological response in vivo, we studied the effects of the biophysically defined pre‐fibrillar αS mutants after expression in tissue culture cells, in mammalian neurons and in PD model organisms, such as Caenorhabditis elegans and Drosophila melanogaster. The results show a striking correlation between αS aggregates with impaired β‐structure, neuronal toxicity and behavioural defects, and they establish a tight link between the biophysical properties of multimeric αS species and their in vivo function.

Fibril structure of amyloid-beta (1-42) by cryo-electron microscopy.

Elucidating pathological fibril structure Amyloid-β (Aβ) is a key pathological contributor to Alzheimers disease. Gremer et al. used cryoelectron microscopy data to build a high-quality de novo atomic model of Aβ fibrils (see the Perspective by Pospich and Raunser). The complete structure reveals all 42 amino acids (including the entire N terminus) and provides a structural basis for understanding the effect of several disease-causing and disease-preventing mutations. The fibril consists of two intertwined protofilaments with an unexpected dimer interface that is different from those proposed previously. The structure has implications for the mechanism of fibril growth and will be an important stepping stone to rational drug design. Science, this issue p. 116; see also p. 45 Cryo–electron microscopy structure of an amyloid-β(1–42) fibril reveals a protofilament interface and the entire N-terminal region. Amyloids are implicated in neurodegenerative diseases. Fibrillar aggregates of the amyloid-β protein (Aβ) are the main component of the senile plaques found in brains of Alzheimer’s disease patients. We present the structure of an Aβ(1–42) fibril composed of two intertwined protofilaments determined by cryo–electron microscopy (cryo-EM) to 4.0-angstrom resolution, complemented by solid-state nuclear magnetic resonance experiments. The backbone of all 42 residues and nearly all side chains are well resolved in the EM density map, including the entire N terminus, which is part of the cross-β structure resulting in an overall “LS”-shaped topology of individual subunits. The dimer interface protects the hydrophobic C termini from the solvent. The characteristic staggering of the nonplanar subunits results in markedly different fibril ends, termed “groove” and “ridge,” leading to different binding pathways on both fibril ends, which has implications for fibril growth.

Solid-state NMR reveals structural differences between fibrils of wild-type and disease-related A53T mutant α-synuclein.

Fibrils from the Parkinsons-disease-related A53T mutant of alpha-synuclein were investigated by solid-state NMR spectroscopy, electron microscopy, and atomic force microscopy. Sequential solid-state NMR resonance assignments were obtained for a large fraction of the fibril core. Experiments conducted above and below the freezing point suggest that the fibrils contain regions with increased mobility and structural elements different from beta-strand character, in addition to the rigid beta-sheet-rich core region. As in earlier studies on wild-type alpha-synuclein, the C-terminus was found to be flexible and unfolded, whereas the main core region was highly rigid and rich in beta-sheets. Compared to fibrils from wild-type alpha-synuclein, the well-ordered beta-sheet region extends to at least L38 and L100. These results demonstrate that a disease-related mutant of alpha-synuclein differs in both aggregation kinetics and fibril structure.

Heck reactions between aryl halides and olefins catalysed by Pd-complexes entrapped into zeolites NaY

Abstract Palladium complexes entrapped into zeolite cages have been prepared and characterised by MAS-NMR. They exhibit a high activity towards the Heck reaction of aryl bromides with olefins for small palladium concentrations (0.1 mol%). The catalysts can be easily separated from the reaction mixture and reused after washing without loss in activity. Except for very large complexes, no limitation to the diffusion of educts in the zeolite cages was observed. As for the homogeneous Heck reaction, the electronic nature of the aryl bromides and the olefins has a dominating effect on the reaction yield and selectivity. The heterogeneous catalysts activate even aryl chlorides under standard reaction conditions.

High‐Resolution Solid‐State NMR Studies on Uniformly [13C,15N]‐Labeled Ubiquitin

Understanding of the effects of intermolecular interactions, molecular dynamics, and sample preparation on high‐resolution magic‐angle spinning NMR data is currently limited. Using the example of a uniformly [13C,15N]‐labeled sample of ubiquitin, we discuss solid‐state NMR methods tailored to the construction of 3D molecular structure and study the influence of solid‐phase protein preparation on solid‐state NMR spectra. A comparative analysis of 13C′, 13Cα, and 13Cβ resonance frequencies suggests that 13C chemical‐shift variations are most likely to occur in protein regions that exhibit an enhanced degree of molecular mobility. Our results can be refined by additional solid‐state NMR techniques and serve as a reference for ongoing efforts to characterize the structure and dynamics of (membrane) proteins, protein complexes, and other biomolecules by high‐resolution solid‐state NMR.

Structural Characterization of Polyglutamine Fibrils by Solid-State NMR Spectroscopy

Protein aggregation via polyglutamine stretches occurs in a number of severe neurodegenerative diseases such as Huntingtons disease. We have investigated fibrillar aggregates of polyglutamine peptides below, at, and above the toxicity limit of around 37 glutamine residues using solid-state NMR and electron microscopy. Experimental data are consistent with a dry fibril core of at least 70-80 Å in width for all constructs. Solid-state NMR dipolar correlation experiments reveal a largely β-strand character of all samples and point to tight interdigitation of hydrogen-bonded glutamine side chains from different sheets. Two approximately equally frequent populations of glutamine residues with distinct sets of chemical shifts are found, consistent with local backbone dihedral angles compensating for β-strand twist or with two distinct sets of side-chain conformations. Peptides comprising 15 glutamine residues are present as single extended β-strands. Data obtained for longer constructs are most compatible with a superpleated arrangement with individual molecules contributing β-strands to more than one sheet and an antiparallel assembly of strands within β-sheets.

Correlation of Amyloid Fibril β-Structure with the Unfolded State of α-Synuclein

Many human neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases, are associated with the deposition of proteinaceous aggregates known as amyloid fibrils. 2] Surprisingly, proteins with very different amino acid sequences and three-dimensional structures aggregate into amyloid fibrils that share common characteristics, such as a similar morphology and a specific b-sheet-based molecular architecture. This suggests that the ability to fibrillate is an intrinsic property of a polypeptide chain and that the native structure is not necessarily the only ordered structure that each protein can assume. An additional common property of aggregation into amyloid fibrils is the presence of partially or fully unfolded states of the misfolding proteins. For proteins that are natively folded, unfolded states are present during biosynthesis, or as a result of proteolytic degradation. Whereas there is increasing knowledge about the factors that drive aggregation, the structural characteristics of aggregation-prone states and the molecular details that determine the arrangement of misfolded proteins in amyloid fibrils are still only understood in outline. Is a high population of a specific conformation in a denatured state required for the efficient generation of amyloid fibrils? What is the relation between the properties of the unfolded state and the b-structure in amyloid fibrils? Are differences in the morphologies of the resulting fibrils associated with differences in the structures of the precursor states? Here we demonstrate by a combination of solution-state and solid-state NMR spectroscopy that the structure of amyloid fibrils is directly correlated to the conformational properties of the unfolded state. Similar or even identical spectroscopic probes are used in solution-state and solidstate NMR, allowing a detailed comparison of the structure of proteins in their unfolded and fibrillar states. a-Synuclein (aS) is the major component of abnormal aggregates in the brains of patients with Parkinson’s disease, the most common neurodegenerative movement disorder. When bound to membranes, the 140-amino acid protein adopts an a-helical conformation. In solution, monomeric aS was found to be highly dynamic and was classified as natively unfolded. We and others recently showed that, despite its high flexibility, native aS adopts an ensemble of conformations stabilized by long-range interactions. Conditions that destabilize long-range interactions lead to conformations that associate readily, resulting in aggregation in vitro. The structure of aS in amyloid fibrils was studied by various techniques, and these studies showed that the cores of aS fibrils are formed by residues ~34 to ~101, comprising at least four bstrands, whereas the Nand C-terminal domains remain disordered. When the temperature of the solution is reduced sufficiently without freezing, proteins can be cold denatured without the need for chemicals that would potentially interfere with the ensemble of conformations present in solution. In addition, ACHTUNGTRENNUNGinterconversion between different conformations that causes averaging of spectroscopic probes at higher temperatures is slowed down (see Figure S1 in the Supporting Information). Here we have used supercooling to remove transient longrange interactions in aS. Using thin glass capillaries, we reduced the temperature in the protein solution to 15 8C without freezing. At 15 8C the hydrodynamic radius of aS was ACHTUNGTRENNUNGsignificantly increased and adopted a value similar to that observed in the presence of 8m urea at 2 8C, 15 8C and 47 8C (Figure 1A). At high temperature, on the other hand, the hydrodynamic radius reached a maximum value that was further increased upon addition of urea, suggesting that long-range ACHTUNGTRENNUNGinteractions are more efficiently removed at 15 8C than at 57 8C. We then determined the sequence-specific assignment of backbone resonances of aS by 3D triple-resonance NMR experiments and observed paramagnetic relaxation enhancement of amide protons due to the presence of a nitroxide spin label attached to residue 18. The reduction in line broadening at 15 8C at the C terminus indicates the removal of transient long-range interactions (Figure S3), in agreement with the increase in hydrodynamic radius. NMR chemical shifts, especially of C and C’ atoms, are very sensitive probes of secondary structure in proteins. These shifts showed small but distinct deviations from random coil values for aS in solution at 15 8C (Figure 1B). For residues 1– 38, the signs of secondary NMR chemical shifts alternated; this indicated the absence of a propensity for either helical or bstructure. Residues 38–98, which form the cores of amyloid fibrils, as well as the acidic C-terminal domain, showed predominantly negative secondary chemical shifts. Whereas negative secondary chemical shifts are characteristic of either b-structure or polyproline helix-like conformations, large JACHTUNGTRENNUNG(H,H) scalar couplings are only found in extended b-structures. In the C-terminal domain of aS, the JACHTUNGTRENNUNG(H,H) values were mostly below average, in agreement with the presence of several proline residues. Only for residues 38–98 were negative secondary chemical shifts and above-average J ACHTUNGTRENNUNG(H,H) couplings observed; this suggests that the central domain of aS transiently populates the b-region in the Ramachandran plot. [a] H.-Y. Kim, Dr. H. Heise, Dr. M. Baldus, Dr. M. Zweckstetter Department of NMR-based Structural Biology Max Planck Institute for Biophysical Chemistry Am Fassberg 11, 37077 Gçttingen (Germany) Fax: (+49)551-201-2202 E-mail : mzwecks@gwdg.de [b] Dr. C. O. Fernandez Instituto de Biolog;a Molecular y Celular de Rosario Universidad Nacional de Rosario Suipacha 531, S2002 LRK Rosario (Argentina) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

Protein solid-state NMR resonance assignments from (13C,13C) correlation spectroscopy

It is demonstrated that sequential resonance assignments can be obtained from (13C,13C) correlation spectroscopy on a uniformly labeled protein under magic angle spinning. The experiment relies on weak (C′,Cα) coupling conditions using a defined range of MAS rates and can be employed at arbitrary magnetic field strength.

Solid‐State NMR Spectroscopy of Amyloid Proteins

Protein misfolding and aggregation is linked to a large number of neurodegenerative and systemic diseases. Prominent examples are the spongiform encephalopathies, Parkinson’s disease, Alzheimer’s disease and Huntington’s disease, and each pathology is accompanied by the aggregation and deposition of one or more particular proteins in various tissues. Furthermore, the phenomenon of a transformation of functional proteins into the infectious prion forms, which leads to strains with altered phenotypes, has been discovered to occur also in yeast cells and fungi. In all these cases, proteins undergo a structural transition from a native globular or unfolded conformation into well-ordered aggregates that are characterised by fibrillar morphology and a high content of b-sheets. A common structural motif to amyloid fibrils is the cross-b pattern, which is defined by b-strands that are perpendicular to the fibril main axis, and hydrogen bonds that are parallel to the fibril axis. Under appropriate conditions, amyloids can be formed by virtually all polypeptides or proteins, independent of the primary sequence. This observation led to the hypothesis that the amyloid conformation is the “primordial” protein structure. Because the amyloid pattern is encoded in the backbone rather than the sequence, polypeptides of a given amino acid sequence can form different polymorphic forms, which are often associated with different fibril morphology, different toxicities, or in the case of prion proteins, they lead to different strains. Besides their physiological relevance as possible disease-causing agents, the structure and aggregation behaviour of amyloid fibrils are also of general interest because they give insight into macromolecular self-organisation and protein folding. Except for short model peptides, amyloid fibrils are intrinsically noncrystalline and insoluble, and are therefore not amenable to high-resolution structure determination techniques such as X-ray crystallography or high-resolution NMR spectroscopy. Low-resolution techniques such as electron microscopy, atomic force microscopy, and X-ray diffraction have yielded valuable information on the morphology and size of amyloid fibrils as well as the common structural element of amyloid fibrils, the cross-b pattern. Furthermore, aggregation studies in combination with site-directed mutagenesis have added valuable constraints on various amyloids. EPR studies of aggregates with spin labels that are attached to single cysteine mutants have placed constrains on defined core regions as well as on the relative orientation of b-strands. Finally, magic angle spinning (MAS) solid-state NMR spectroscopy has become a powerful tool for structural studies on immobilised proteins and protein complexes, and it has been shown to be the ideal tool for the investigation of amyloid fibrils from short model peptides as well as from full-length proteins. In this review, an overview of solid-state NMR spectroscopic studies on amyloid fibrils and a review of selected recent results is presented.

Interaction of Epothilone B (Patupilone) with Microtubules as Detected by Two‐Dimensional Solid‐State NMR Spectroscopy

Solid evidence: Induction of the polymerization of β-tubulin dimers into microtubules by epothilones, such as patupilone, by an as yet unknown mechanism leads to the apoptosis of cancer cells. Solid-state NMR spectroscopy of patupilone bound to microtubules has now enabled the identification of atomic positions of the drug that undergo clear chemical-shift changes upon binding (see correlation spectra of free (black) and complexed patupilone (red)).