One Sentence Summary: Oligomers of α-synuclein generate neuronal damage when insertion of a highly structured core causes disruption of membrane integrity. This manuscript has been accepted for publication in Science. This version has not undergone final editing. Please refer to the complete version of record at http://www.sciencemag.org/. The manuscript may not be reproduced or used in any manner that does not fall within the fair use provisions of the Copyright Act without the prior, written permission of AAASAbstract: Oligomeric species populated during the aggregation process of α-synuclein have been linked to neuronal impairment in Parkinson's disease and related neurodegenerative disorders. By using solution and solid-state NMR techniques in conjunction with other structural methods, we identified the fundamental characteristics that enable toxic α-synuclein oligomers to perturb biological membranes and disrupt cellular function; these include a highly lipophilic element that promotes strong membrane interactions and a structured region that inserts into lipid bilayers and disrupts their integrity. In support of these conclusions, mutations that target the region that promotes strong membrane interactions by α-synuclein oligomers suppressed their toxicity in neuroblastoma cells and in primary cortical neurons.

We have studied two misfolded oligomeric forms of the protein HypF-N, which show similar morphologies but very different toxicities. We measured over 80 intermolecular distance-dependent parameters for each oligomer type using FRET, in conjunction with solution- and solid-state NMR and other biophysical techniques. The results indicate that the formation of a highly organised hydrogen bonded core in the toxic oligomers results in the exposure of a larger number of hydrophobic residues than in the nontoxic species, causing the former to form aberrant interactions with cellular components.

There continues to be a significant increase in the number and complexity of hydrophobic nanomaterials that are engineered for a variety of commercial purposes making human exposure a significant health concern. This study uses a combination of biophysical, biochemical and computational methods to probe potential mechanisms for uptake of C60 nanoparticles into various compartments of living immune cells. Cultures of RAW 264.7 immortalized murine macrophage were used as a canonical model of immune-competent cells that are likely to provide the first line of defense following inhalation. Modes of entry studied were endocytosis/pinocytosis and passive permeation of cellular membranes. The evidence suggests marginal uptake of C60 clusters is achieved through endocytosis/pinocytosis, and that passive diffusion into membranes provides a significant source of biologically-available nanomaterial. Computational modeling of both a single molecule and a small cluster of fullerenes predicts that low concentrations of fullerenes enter the membrane individually and produce limited perturbation; however, at higher concentrations the clusters in the membrane causes deformation of the membrane. These findings are bolstered by nuclear magnetic resonance (NMR) of model membranes that reveal deformation of the cell membrane upon exposure to high concentrations of fullerenes. The atomistic and NMR models fail to explain escape of the particle out of biological membranes, but are limited to idealized systems that do not completely recapitulate the complexity of cell membranes. The surprising contribution of passive modes of cellular entry provides new avenues for toxicological research that go beyond the pharmacological inhibition of bulk transport systems such as pinocytosis.

An efficient NMR methods for the characterisation of 14N sites has been developed with efficiencies suitable for the quantitative analysis of biomolecular and natural abundance systems.

Overtone 14N NMR spectroscopy is a promising route for the direct detection of 14N signals with good spectral resolution.