The Application of NMR to Study the Parkinson's Protein Alpha-Synuclein

Abstract

α-Synuclein (αS) is a 14 kDa intrinsically disordered protein found in the substantia nigra
region of the brain. αS has high conformational plasticity, and misfolding of αS to form
amyloid fibrils is a hallmark of Parkinson’s disease (PD) pathology. It is widely acknowledged
that the physiological function of αS includes lipid binding, and lipid-induced oligomerisation
is thought to be a key event in PD. Therefore, elucidation of the structure and behaviour of αS
in the presence of lipid membranes is critical to identifying a viable therapeutic target for PD
treatment.
Nuclear magnetic resonance (NMR) is an indispensable tool for studying intrinsically
disordered proteins (IDPs) as it is one of the few tools able to study IDPs in close to
physiological conditions with atomic-level resolution. The use of lipids to study αS, however,
poses several challenges for solution NMR. Conversely, the application of co-solvents, e.g.
2,2,2-trifluoroethanol (TFE), might offer a route to access folded states of αS similar to those
induced by lipid vesicles whilst avoiding the associated complications.
Here, the NMR and biophysical characterisation of αS in the presence of various lipid
mimetics, including TFE, is presented. NMR chemical shift and structural data of αS in 50%
TFE was collected and analysed. Together, these demonstrate that globally, αS forms two
distinct domains, an α-helical domain spanning residues 3-98 and an unstructured domain
spanning residues 99-140, in agreement with lipid-bound structural studies. Examination of
15N relaxation data revealed that regions of reduced helicity in residues 3-98 were also
potentially captured. Overall, the TFE co-solvent appeared to act as a robust model for αS in a
lipid environment.
Using this model co-solvent, the interaction of two peptides, αS2-12(L6) and αS2-12(W), with αS
was explored. Developed by collaborators at the University of Bath, these were found to inhibit
αS aggregation in a dose-dependent manner. Chemical shift perturbations identified two
binding regions for the peptides on αS. The first in the C-terminus and the second in the
preNAC region, which is only observed when αS is in the α-helical conformation induced by
50% TFE. Additionally, 2D heteronuclear NMR confirmed that αS2-12(L6) stabilises
monomeric αS while it seems αS2-12(W) must stabilise oligomeric species beyond the size limit
of NMR. These results suggest that although the peptides appear to target similar regions of
αS, they likely have different mechanisms of action.

Date of Award	10 Dec 2024
Original language	English
Awarding Institution	University of Bristol
Supervisor	Matthew P Crump (Supervisor) & Jody M. Mason (Supervisor)