James A. Jarvis

Structural basis of membrane disruption and cellular toxicity by α-synuclein oligomers

A structural look at α-synuclein oligomers Fibrillar aggregates of the protein α-synuclein (αS) are the major constituents of Lewy bodies in Parkinsons disease. However, small oligomers that accumulate during the process of fibril formation are thought to cause the neuronal toxicity associated with the onset and progression of Parkinsons disease. Little is known about the detailed structural properties of αS oligomers and the molecular mechanisms that lead to their toxicity. Fusco et al. report the structural characterization of two forms of αS oligomers, which elucidates the fundamental structural elements giving rise to neuronal toxicity. Science, this issue p. 1440 Oligomers of α-synuclein generate neuronal damage when insertion of a highly structured core disrupts membrane integrity. 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 nuclear magnetic resonance 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 primary cortical neurons.

14 N overtone NMR under MAS: signal enhancement using symmetry-based sequences and novel simulation strategies†

Overtone (14)N NMR spectroscopy is a promising route for the direct detection of (14)N signals with good spectral resolution. Its application is currently limited, however, by the absence of efficient polarization techniques for overtone signal enhancement and the lack of efficient numerical simulation techniques to aid in both the development of new methods and the analysis and interpretation of experimental data. In this paper we report a novel method for the transfer of polarization from (1)H to the (14)N overtone using symmetry-based R-sequences that overcome many of the limitations of adiabatic approaches that have worked successfully on static samples. Refinement of these sequences and the analysis of the resulting spectra have been facilitated through the development of an efficient simulation strategy for (14)N overtone NMR spectroscopy of spinning samples, using effective Hamiltonians on top of Floquet and Fokker-Planck equations.Overtone 14N NMR spectroscopy is a promising route for the direct detection of 14N signals with good spectral resolution.

An efficient NMR method for the characterisation of 14N sites through indirect 13C detection

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.

C60 fullerene localization and membrane interactions in RAW 264.7 immortalized mouse macrophages

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.

CHAPTER 5:Membrane Protein Interactions

Integral membrane proteins support a number of important cellular processes playing a key role in the transport of information and material across cellular membranes. Indeed, miss-regulation or miss-localization of these proteins has been linked to several important disease states. It has become abundantly clear that the activity of these proteins and their localization within the cell critically depends on their interactions with other cellular components, including small molecules destined for translocation across the bilayer or involved in cell signalling, lipids present within the lipid bilayer and other integral and soluble proteins. Accordingly, if we are to understand how integral membrane proteins fulfill these important roles it is vital we understand how they interact with their local cellular environment at the molecular level. Solid-state NMR has emerged as a powerful tool to study how integral membrane proteins interact with other cellular partners, facilitating the study of these interactions at the molecular level. Importantly, as we highlight, these processes can now be studied whilst the protein is resident within the lipid bilayer, which provides unique insights into how interactions between the lipid bilayer and integral membrane proteins can play an important role in regulating vital cellular and disease related processes.