Unravelling the Cause of Parkinson's Disease in Molecular Detail

A-Synuclein is a 140 amino acid, 14 kDa, intrinsically
disordered protein. Neuronal aggregates of a-Synuclein are
the hallmarks of Parkinson's Disease. The protein contains
a series of imperfect KTKEGV motifs carrying a net positive
charge spread across the first 25 N-terminal residues and
central NAC region, covering residues 25-100 whilst the Cterminal is negatively charged. NMR chemical shift
assignments of the monomer in solution have been
published. A-Synuclein associates with lipid membrane
surfaces in a sequential manner, with an N-terminal anchor
strongly bound and the NAC membrane sensing region less
strongly bound. On binding, both these regions adopt an
amphipathic helical conformation. Exposure of the
membrane bound NAC region has been implicated in
aberrant misfolding, oligomerisation, aggregation,
membrane insertion and pore formation. A-Synuclein
oligomers are highly toxic to cells and act as seeding
species in intercellular spread. Solid state and Cryo-EM
studies have revealed the structure of the end stage fibrils
but mechanistic studies of the membrane bound monomer
to oligomer transition have proven challenging although the
NMR solution structure of a-Synuclein bound to an SLS
micelle has been published.
In this proposal we will exploit the small size of detergent
micelles to facilitate acquisition of high resolution solution
state NMR data. In previous studies a-Synuclein has been
uniformly labelled with NMR active nuclei. Here we will
employ a selective labelling approach using Cysteine point
mutations to install NMR active labels at strategic points
distributed along the linear sequence of the protein. This
will allow us to visualise in molecular detail the assembly
of a-Synuclein molecules in a way not possible with
uniformly labelled material due to severe spectroscopic
overlap when multiple copies of protein are present. We
will confirm the integrity of the modified Synucleins with
standard biophysical analysis. We will generate a panel of
single and doubly labelled proteins suitable for inter and
intra-molecular nOe experiments revealing the mechanism
of the earliest events in dimer, trimer, tetramer and high
order oligomer formation and their morphology viz:
parallel, antiparallel, in or out of register.
Further we will develop customised NMR probes of different
vector length and orientation providing data on both short
and long range interactions in the lipid bound state.
Finally we will apply our approach to naturally occurring
familial mutant a-Synucleins known to have differentiated
membrane binding affinity which is correlated with disease
pathology in terms of onset of age and rate of progression.