Structure and dynamics of oligomeric intermediates in amyloid assembly

淀粉样变与阿尔茨海默氏症、帕金森症和II型糖尿病等毁灭性疾病有关。尽管导致这些疾病的蛋白质在氨基酸序列和单体前体结构上差异很大，但它们都形成了称为淀粉样蛋白原纤维的不溶性聚合物结构，这些结构与所谓的交叉结构具有共同的独特形态。许多蛋白质的组装途径被认为是从蛋白质单体部分或完全从其特定的天然3D结构展开开始的。一旦展开，该单体能够聚合或自组装，形成独特的、长而扭曲的原纤维，这些原纤维积聚在身体的各个器官中，与淀粉样蛋白疾病有关。然而，组装的精确途径和寡聚中间体的性质（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，我们建议以前所未有的细节来表征这些低聚物，从而在分子细节上绘制淀粉样变性的途径，并为未来的治疗方法确定可能的低聚物靶点。为了解决这些问题，我们将研究淀粉样蛋白β -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原纤维的蛋白质。申请人在过去几年中已经获得了丰富的这种蛋白质的经验，因此它是进行拟议研究的理想系统。除了对beta2m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。为了全面描绘淀粉样蛋白组装途径，我们建议采用一种新的和令人兴奋的两种技术组合：离子迁移谱法和质谱法。在一个单一的、快速的实验中，我们现在能够在纤维组装过程中实时检测、量化和单独表征异质系中的瞬态中间体。基于证明这种方法的能力的大量初步数据，我们将测量(i)分子质量，（ii）横截面积（由形状决定），（iii）稳定性（使用碰撞诱导解离和亚基交换实验）和（iv）不同的单个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模拟程序来比较实验数据和理论上合理的结构。通过将这些实验与蛋白质工程相结合，其中单个氨基酸的侧链将一个接一个地改变，以及其他生化和生物物理分析，我们将确定哪些残基负责特定低聚物的形成，从而绘制出每种低聚物在原纤维组装中的重要性。我们还将使用所开发的方法来比较生理和非生理条件下的组装，以辨别组装景观的异质性。最后，我们将研究一种小分子配体的结合，我们最近发现这种配体是一种新型的、有效的β - 2淀粉样蛋白组装抑制剂。将评估配体结合对单个寡聚物种的种群，结构和稳定性的影响，为小配体如何全面阻止大蛋白质亚基的自组装提供新的见解。总之，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，并将大大提高我们对这种不必要的生物现象的理解。此外，通过表征单个低聚物的结构和配体结合特性，我们的目标是首次确定单个分子物种作为未来治疗干预的目标。

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.