Unravelling the Cause of Parkinson's Disease in Molecular Detail

A-突触核蛋白是一种由140个氨基酸组成的14 kDa的固有无序蛋白质。α-突触核蛋白的神经元聚集是帕金森氏症的特征。该蛋白含有一系列不完整的KTKEGV基序，携带一个净正电荷分布在前25个N末端残基和中央NAC区域，覆盖25-100个残基，而CTeral带负电荷。对单体在溶液中的核磁共振化学位移进行了指认。α-突触核蛋白与脂质膜表面以顺序的方式结合，N-末端锚定强结合，NAC膜敏感区结合较弱。在结合时，这两个区域都采用非两亲性螺旋构象。膜结合的NAC区域的暴露涉及到异常的错误折叠、寡聚、聚集、膜插入和孔形成。α-突触核蛋白低聚物对细胞有很强的毒性，在细胞间扩散中充当种子物种。固态和低温EM研究揭示了终末纤维的结构，但膜结合单体到低聚物转变的机理研究被证明是具有挑战性的，尽管已经发表了结合到SLS胶束上的α-突触核蛋白的核磁共振溶液结构。在这个建议中，我们将利用洗涤剂胶束的小尺寸来促进高分辨率溶液状态核磁共振数据的获取。在以前的研究中，α-突触核蛋白被核磁共振活性核均匀标记。在这里，我们将使用半胱氨酸点突变的选择性标记方法，将核磁共振活性标记安装在沿蛋白质线性序列分布的策略点。这将使我们能够在分子细节上可视化a-突触核蛋白分子的组装，而这种方式是不可能用统一标记的材料的，因为当存在多个蛋白质副本时，会有严重的光谱重叠。我们将通过标准的生物物理分析来确认修饰后的突触核蛋白的完整性。我们将产生一组适用于分子间和分子内NOE实验的单标记和双标记蛋白质，揭示二聚体、三聚体、四聚体和高阶低聚物形成的最早事件的机制及其形态：平行、反平行，登记内或登记外。此外，我们将开发不同载体长度和取向的定制核磁共振探针，提供脂结合状态中短距离和长距离相互作用的数据。最后，我们将应用我们的方法来研究已知具有不同膜结合亲和力的自然发生的家族性突变型a-突变体，这与疾病病理学的发病年龄和进展速度相关。

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.