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.

The 140-residue protein ␣-synuclein (AS) is able to form amyloid fibrils and as such is the main component of protein inclusions involved in Parkinson's disease. We have investigated the structure and dynamics of full-length AS fibrils by high-resolution solid-state NMR spectroscopy. Homonuclear and heteronuclear 2D and 3D spectra of fibrils grown from uniformly 13 C͞ 15 N-labeled AS and AS reverse-labeled for two of the most abundant amino acids, K and V, were analyzed. 13 C and 15 N signals exhibited linewidths of <0.7 ppm. Sequential assignments were obtained for 48 residues in the hydrophobic core region. We identified two different types of fibrils displaying chemical-shift differences of up to 13 ppm in the 15 N dimension and up to 5 ppm for backbone and side-chain 13 C chemical shifts. EM studies suggested that molecular structure is correlated with fibril morphology. Investigation of the secondary structure revealed that most amino acids of the core region belong to ␤-strands with similar torsion angles in both conformations. Selection of regions with different mobility indicated the existence of monomers in the sample and allowed the identification of mobile segments of the protein within the fibril in the presence of monomeric protein. At least 35 C-terminal residues were mobile and lacked a defined secondary structure, whereas the N terminus was rigid starting from residue 22. Our findings agree well with the overall picture obtained with other methods and provide insight into the amyloid fibril structure and dynamics with residue-specific resolution.EM ͉ protein structure ͉ amyloid ͉ Parkinson's disease ͉ protein aggregation T he ability of a protein to form amyloid fibrils is increasingly recognized as a general property of all polypeptide sequences (1). It is a phenomenon common to numerous neurodegenerative diseases such as Alzheimer's and Parkinson's diseases and spongiform encephalopathies (2, 3). Thus, the investigation of the mechanism(s) of protein misfolding as well as the detailed structures of amyloid fibrils is of paramount interest (4, 5). Parkinson's disease is defined by the presence of intracellular inclusions in dopaminergic neurons, the so-called Lewy bodies, which contain a high content of fibrils formed from the 140-aa cytoplasmic protein ␣-synuclein (AS) (6). The question of whether the mature fibrils themselves or rather protofilaments or folding intermediates are the neurotoxic species responsible for the cell death of dopaminergic neurons is still a subject of great controversy (7-9).AS belongs to the class of natively unfolded proteins, i.e., it lacks a well-defined secondary structure (10, 11), although long-range interactions have been shown to stabilize an aggregation-autoinhibited global protein architecture (12). Three regions of the protein can be classified. (i) The amphipathic N terminus (residues 1-60) consists of imperfect 11-mer repeats, with the consensus motif KTKEGV. (ii) The predominantly hydrophobic middle, also known as non-A␤ component region (residues 61-95...

The relation of a-synuclein (aS) aggregation to Parkinson's 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 aS species have in neurotoxicity in vivo, we generated aS variants by a structurebased rational design. Biophysical analysis revealed that the aS 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 aS 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 aS aggregates with impaired b-structure, neuronal toxicity and behavioural defects, and they establish a tight link between the biophysical properties of multimeric aS species and their in vivo function.

It is shown that molecular structure and dynamics of a uniformly labeled membrane protein can be studied under magic-angle-spinning conditions. For this purpose, dipolar recoupling experiments are combined with novel through-bond correlation schemes that probe mobile protein segments. These NMR schemes are demonstrated on a uniformly [13C,15N] variant of the 52-residue polypeptide phospholamban. When reconstituted in lipid bilayers, the NMR data are consistent with an alpha-helical trans-membrane segment and a cytoplasmic domain that exhibits a high degree of structural disorder.