Unravelling the Cause of Parkinson's Disease in Molecular Detail

点击翻译按钮获取中文摘要

A-Synuclein is a 140 amino acid, 14 kDa, intrinsicallydisordered protein. Neuronal aggregates of a-Synuclein arethe hallmarks of Parkinson's Disease. The protein containsa series of imperfect KTKEGV motifs carrying a net positivecharge spread across the first 25 N-terminal residues andcentral NAC region, covering residues 25-100 whilst the Cterminal is negatively charged. NMR chemical shiftassignments of the monomer in solution have beenpublished. A-Synuclein associates with lipid membranesurfaces in a sequential manner, with an N-terminal anchorstrongly bound and the NAC membrane sensing region lessstrongly bound. On binding, both these regions adopt anamphipathic helical conformation. Exposure of themembrane bound NAC region has been implicated inaberrant misfolding, oligomerisation, aggregation,membrane insertion and pore formation. A-Synucleinoligomers are highly toxic to cells and act as seedingspecies in intercellular spread. Solid state and Cryo-EMstudies have revealed the structure of the end stage fibrilsbut mechanistic studies of the membrane bound monomerto oligomer transition have proven challenging although theNMR solution structure of a-Synuclein bound to an SLSmicelle has been published.In this proposal we will exploit the small size of detergentmicelles to facilitate acquisition of high resolution solutionstate NMR data. In previous studies a-Synuclein has beenuniformly labelled with NMR active nuclei. Here we willemploy a selective labelling approach using Cysteine pointmutations to install NMR active labels at strategic pointsdistributed along the linear sequence of the protein. Thiswill allow us to visualise in molecular detail the assemblyof a-Synuclein molecules in a way not possible withuniformly labelled material due to severe spectroscopicoverlap when multiple copies of protein are present. Wewill confirm the integrity of the modified Synucleins withstandard biophysical analysis. We will generate a panel ofsingle and doubly labelled proteins suitable for inter andintra-molecular nOe experiments revealing the mechanismof the earliest events in dimer, trimer, tetramer and highorder oligomer formation and their morphology viz:parallel, antiparallel, in or out of register.Further we will develop customised NMR probes of differentvector length and orientation providing data on both shortand long range interactions in the lipid bound state.Finally we will apply our approach to naturally occurringfamilial mutant a-Synucleins known to have differentiatedmembrane binding affinity which is correlated with diseasepathology in terms of onset of age and rate of progression.