Structure and dynamics of oligomeric intermediates in amyloid assembly

淀粉样变性与包括阿尔茨海默氏症、帕金森氏症和II型糖尿病在内的毁灭性疾病有关。尽管导致这些疾病的蛋白质在其氨基酸序列和单体前体的结构方面差异很大，但它们都形成称为淀粉样蛋白原纤维的不溶性聚合物结构，其具有共同的独特形态，具有所谓的交叉β结构。许多蛋白质的组装途径被认为始于蛋白质单体部分或完全从其特定的天然3D结构展开。一旦展开，单体就能够聚合或自组装成独特的、长的、扭曲的原纤维，这些原纤维积聚在身体的各个器官中，并与淀粉样蛋白疾病有关。然而，组装的精确途径和低聚中间体的性质（其中一种或多种被认为是与淀粉样蛋白疾病相关的毒性的罪魁祸首）仍然未知。在这里，我们建议在前所未有的细节，这些寡聚体，从而映射淀粉样变性的分子细节的途径，并确定未来的治疗措施可能的寡聚体的目标。为了解决这些问题，我们将研究淀粉样蛋白，β 2-微球蛋白（β 2m），一种在所有接受长期肾透析的患者中形成淀粉样纤维的蛋白质。申请人在几年内已经获得了这种蛋白质的丰富经验，因此它是进行所提出的研究的理想系统。除了对β 2m原纤维组装的新见解外，所产生的结果和开发的方案将对所有淀粉样蛋白系统具有直接的重要性和相关性。为了描绘淀粉样蛋白组装途径的全面图景，我们建议采用两种技术的新的和令人兴奋的组合：离子迁移谱和质谱。在一个单一的，快速的实验中，我们现在能够检测，定量和单独的瞬时中间体内的异质合奏实时原纤维组装。建立在大量的初步数据，证明这种方法的权力，我们将测量（i）的分子量，（ii）的横截面积（由形状决定），（iii）的稳定性（使用碰撞诱导解离和亚基交换实验）和（iv）不同的，个别的寡聚物物种的配体结合能力。我们还将使用内部开发的分子建模程序来比较实验数据与理论上合理的结构。通过将这些实验与蛋白质工程（其中单个氨基酸的侧链将被逐个改变）以及其他生物化学和生物物理分析相结合，我们将确定哪些残基是形成特定寡聚体的原因，从而绘制出每个寡聚体在原纤维组装中的重要性。我们还将使用开发的方法来比较生理和非生理条件下的组装，以便辨别组装景观的异质性。最后，我们将研究一个小分子配体的结合，我们最近确定为新的，有效的β 2m淀粉样蛋白组装抑制剂。配体结合的人口，结构和稳定性的个别寡聚物种的后果将进行评估，提供新的见解如何一个小的配体可以全面逮捕一个大的蛋白质亚基的自组装。这些实验将为淀粉样蛋白组装的基本机制提供前所未有的细节，并将大大增强我们对这种不必要的生物现象的理解。此外，通过表征单个寡聚体的结构和配体结合特性，我们的目标是首次确定单个分子种类作为未来治疗干预的靶点。

Amyloidosis is associated with devastating diseases including Alzheimer's, Parkinson's and type II diabetes. Although the proteins responsible for these diseases vary widely in their amino acid sequences and structure of the monomeric precursor, they all form insoluble polymeric structures termed amyloid fibrils, which share a common, distinctive morphology with a so-called cross-beta structure. The assembly pathway for many proteins is thought to start with the protein monomer unfolding partially or completely from its specific, native 3D structure. Once unfolded, the monomer is able to polymerise, or self-assemble, into the distinctive, long, twisted fibrils that accumulate in various organs of the body and are associated with amyloid disease. However, the precise pathways of assembly, and the nature of oligomeric intermediates, (one or more of which are thought to be the culprits of the toxicity associated with amyloid disease), remain unknown. Here we propose to characterise these oligomers in unprecedented detail, thus mapping the pathway of amyloidosis in molecular detail and identifying possible oligomeric targets for future therapeutic remedies. To address these issues, we will work on the amyloidogenic protein, beta2-microglobulin (beta2m), a protein which forms amyloid fibrils in all patients undergoing long term renal dialysis. The applicants have gained a wealth of experience with this protein over several years and hence it is an ideal system on which to carry out the proposed research. In addition to new insights into beta2m fibril assembly, the results generated and protocols developed will have direct importance and relevance for all amyloid systems. To paint a comprehensive picture of the amyloid assembly pathway we propose to employ a new and exciting combination of two techniques: ion mobility spectrometry and mass spectrometry. In a single, rapid experiment we are now able to detect, quantify and individually characterise transient intermediates within heterogeneous ensembles in real-time during fibril assembly. Building on a mass of preliminary data that demonstrate the powers of this approach we will measure (i) the molecular mass, (ii) the cross-sectional area (which is determined by shape), (iii) the stability (using collision-induced dissociation and subunit exchange experiments) and (iv) the ligand binding capability of different, individual oligomeric species. We will also use in-house developed molecular modelling programs to compare the experimental data with theoretically plausible structures. By combining these experiments with protein engineering, in which the side-chains of individual amino acids will be altered one by one, and other biochemical and biophysical analyses, we will determine which residues are responsible for specific oligomers being formed, and will thereby map the importance of each oligomer in fibril assembly. We will also use the methods developed to compare assembly under physiological and non-physiological conditions, so as to discern the heterogeneity of the assembly landscape. Finally, we will study the binding of a small molecule ligand that we have recently identified as novel, potent inhibitor of beta2m amyloid assembly. The consequence of ligand binding on the population, structure, and stability of individual oligomeric species will be assessed, providing new insights into how a small ligand can comprehensively arrest the self-assembly of a large protein subunit. Together these experiments will provide unprecedented detail into the fundamental mechanisms of amyloid assembly and will greatly enhance our understanding of this unwanted biological phenomenon. Moreover, by characterising the structural and ligand binding properties of individual oligomers, we aim to identify, for the first time, individual molecular species as the targets for future therapeutic intervention.