Characterisation of Amyloid- B and its Interactions by NMR Exchange Techniques

阿尔茨海默病是一种致命的神经疾病，是全球痴呆症的主要原因。它的特征是聚集肽，淀粉样蛋白B(AB)的沉积，在大脑中形成斑块。AB通过三种状态转变为聚集态：单体(其中多肽本质上是无序的)、寡聚体(小聚集体，被认为是毒性的主要候选者)和高度有序的腓骨结构。目前大多数药物发现策略的目标是聚集的物种，包括低聚物和纤维。淀粉样前体蛋白被蛋白水解物切割成抗体。40个残基的变异体Ab40是生物体体液中的主要形式。相比之下，42个残基的变体AB42在阿尔茨海默病斑块中更常见，而且有更高的聚集倾向。这两种多肽之间最显著的聚集率差异是在聚集的第一步，即初级成核，这表明单体AB40和AB42存在根本差异。众所周知，这些多肽的单体形式本质上是无序的，但对这两种多肽的结构研究发现，它们在单体状态下的化学或结构差异很小。许多人认为，聚集倾向的差异是由AB42中额外的C-末端疏水氨基酸驱动的，或者是由于低聚物和纤维状物种的结构不同，聚集速率不同。在我3个月的轮换中，我使用了CLEAN-EX核磁共振方法来了解AB40和AB42在溶剂可及性方面的差异。我的初步数据表明，这两个多肽之间的溶剂化速率存在差异，不仅在预期的多肽的C末端，而且在上游残基(K16)上也有差异，后者在AB42中受到的保护令人惊讶。这一观察表明，AB42中额外的氨基酸可能会引起一些长期影响，影响肽的整体结构。在我的博士学位期间，我将首先重复这些干净的前实验来证实我的发现，然后扩展这种方法来更深入地研究AB的单体性质。已经表明，对K16残基(和其他残基)上的AB40进行翻译后修饰会导致多肽的不同聚集谱，如果通过CLEAN-EX实验在单体水平上检测到这种差异，将是有趣的。我计划探索的另一种方法涉及将类似药物的小分子与AB结合。我将首先进行聚集分析，确定改变AB聚集轮廓的分子，然后进行核磁共振实验，以确定单体的结合状态和非结合状态之间的溶剂化是否存在差异。总体而言，关于单体AB的结构还有许多悬而未决的问题，基于溶剂的核磁共振方法将是研究这些问题的一种有趣的、有希望有利可图的方法。

Alzheimer's disease is a fatal neurological condition and the leading cause of dementia worldwide. It is characterised by deposits of aggregated peptide, amyloid-B (AB), which form plaques in the brain. AB transitions into aggregated species through three states: monomeric (where the peptide is intrinsically disordered), oligomeric (small aggregates, identified as the primary candidate for toxicity) and highly ordered fibular structures. Most current drug discovery strategies aim to target the aggregated species including oligomers and fibrils. Amyloid precursor protein is proteolytically cleaved into Ab peptides. The 40-residue variant, Ab40, is the predominant form found in biological fluids. In contrast, the 42-residue variant, Ab42, is more commonly found in Alzheimer's disease plaques and has a higher propensity for aggregation. The most striking difference in aggregation rates between the two peptides is found in the first step of aggregation called primary nucleation, suggesting that there are fundamental differences in monomeric AB40 and AB42. It is well known that these peptides are intrinsically disordered in their monomeric forms, but structural studies of the two peptides have found very little chemical or structural differences in their monomeric states. Many suggest that the difference in aggregation propensity is driven by the additional C-terminal hydrophobic amino acids in AB42 or that the aggregation rates differ due to varying structures of oligomeric and fibrillar species. In my 3-month rotation I used CLEAN-EX NMR methods to understand the difference in solvent accessibility between AB40 and AB42. My preliminary data suggests that there is a difference in the solvation rates between the two peptides, not only at the expected C-terminal end of the peptide, but also at an upstream residue (K16) which is surprisingly more protected in AB42. This observation suggests that the additional amino acids in AB42 may be causing some long-range effects that affect the overall structure of the peptide.During my PhD, I will first repeat these CLEAN-EX experiments to confirm my findings and then extend this approach to delve deeper into the monomeric properties of AB. It has been shown that post translationally modifying AB40 on residue K16 (and other residues) results in a different aggregation profile for the peptide and it would be interesting to see if this difference can be detected at the monomer level by CLEAN-EX experiments. Another avenue I plan to explore involves the binding of small, drug-like molecules to AB. I will first conduct aggregation assays identify molecules that alter the aggregation profile of AB and then follow that up with NMR experiments to identify if there are differences in solvation between the bound and unbound states of the monomer. Overall, there are a lot of unanswered questions about the structure of monomeric AB and solvent-based NMR methods would be an interesting and hopefully lucrative method of investigating these questions.