Biomolecular design of DNA aptamers to target α-Synuclein oligomers in Parkinson’s disease - Metal interactions of α-Synuclein probed by NMR amide-proton exchange

Abstract

α-Synuclein is a small, monomeric intrinsically disordered protein mainly expressed at the presynaptic terminal of neurons. By establishing interactions with neuronal and synaptic vesicle membranes, α-Synuclein is believed to be a critical component in neurotransmission. Under certain conditions, however, the aberrant misfolding and aggregation of α-Synuclein may be triggered, which are strongly linked to the pathology of Parkinson’s disease.

Recent findings have identified the disruption of biological membranes as the main underlying pathological mechanism. Essential structural knowledge of the membrane binding ability of α-Synuclein fundamental to the mechanism of toxicity is exploited in this thesis, with the aim of selecting DNA aptamers targeted at the key N-terminal region to block the detrimental interactions of α-Synuclein oligomeric species with lipid membranes. Identified aptamers, however, failed at showing preferential binding to α-Synuclein at different biophysical assays. Instead, sequences with affinity for streptavidin, which was used to immobilise the target, were unintentionally selected for and isolated.

It is now established that α-Synuclein interacts with a variety of metal ions, which may alter its conformational properties and promote aggregation. Abnormal metal ion levels in neurons may therefore directly contribute to the development of idiopathic Parkinson’s disease by shifting the conformational ensemble of α-Synuclein through the disruption of native intramolecular interactions.

To characterise the nature of these conformational changes, nuclear magnetic resonance was used to study hydrogen exchange of the backbone amide protons as a measure of solvent exposure. These experiments were complemented with chemical shift perturbation and 15N relaxation measurements to obtain a comprehensive view of the interaction between α-Synuclein and the pathophysiologically relevant metal ions Ca2+, Cu2+, Cu+, Mn2+ and Zn2+. Overall, the data presented in this thesis indicate that binding of different metals may induce effects of different structural nature that have the potential of skewing the conformational ensemble towards aggregation-prone conformers.