[论文解读] Conformational equilibria in monomeric alpha-synuclein at the single molecule level

Massimo Sandal, Francesco Valle+7 more|ArXiv.org|Dec 12, 2007

Neurological disorders and treatments参考文献 55被引用 36

一句话总结

本研究利用单分子力谱技术揭示，在生理条件下，单体α-突触核蛋白在无规卷曲、无序和‘β样’结构之间存在动态构象平衡。研究人员发现，与聚集直接相关的‘β样’构象在促进纤维形成的条件下（如Cu2+存在、A30P突变、高离子强度）显著增加，表明纤维形成受化学平衡调控，且该平衡可作为帕金森病治疗的上游靶点。

ABSTRACT

Natively unstructured proteins defy the classical "one sequence-one structure" paradigm of protein science. Monomers of these proteins in pathological conditions can aggregate in the cell, a process that underlies socially relevant neurodegenerative diseases such as Alzheimer and Parkinson. A full comprehension of the formation and structure of the so-called misfolded intermediates from which the aggregated states ensue is still lacking. We characterized the folding and the conformational diversity of alpha-synuclein (aSyn), a natively unstructured protein involved in Parkinson disease, by mechanically stretching single molecules of this protein and recording their mechanical properties. These experiments permitted us to directly observe directly and quantify three main classes of conformations that, under in vitro physiological conditions, exist simultaneously in the aSyn sample, including disordered and "beta-like" structures. We found that this class of "beta-like" structures is directly related to aSyn aggregation. In fact, their relative abundance increases drastically in three different conditions known to promote the formation of aSyn fibrils: the presence of Cu2+, the occurrence of the pathogenic A30P mutation, and high ionic strength. We expect that a critical concentration of aSyn with a "beta-like" structure must be reached to trigger fibril formation. This critical concentration is therefore controlled by a chemical equilibrium. Novel pharmacological strategies can now be tailored to act upstream, before the aggregation process ensues, by targeting this equilibrium. To this end, Single Molecule Force Spectroscopy can be an effective tool to tailor and test new pharmacological agents.

研究动机与目标

• 在单分子水平上理解单体α-突触核蛋白的构象多样性。
• 确定在生理条件下溶液中存在的结构状态。
• 研究与疾病相关的因素（如Cu2+存在、A30P突变、高离子强度）如何影响α-突触核蛋白的构象平衡。
• 识别α-突触核蛋白纤维形成的结构前体。
• 探索靶向构象平衡作为帕金森病新型治疗策略的潜力。

提出的方法

• 采用单分子力谱（SMFS）技术对单个α-突触核蛋白分子进行机械拉伸。
• 记录力-伸展曲线，以探测单个蛋白质的机械稳定性和结构转变。
• 基于机械响应模式，识别出三种不同的构象类别：无序、无规卷曲和‘β样’。
• 在不同条件下量化每种构象的相对丰度：生理缓冲液、含Cu2+、含A30P突变、高离子强度。
• 通过力-伸展数据的统计分析，实现对结构状态的分类并测量其丰度。
• 本研究利用定制的微流控系统，确保在生理条件下实现受控的单分子测量。

实验结果

研究问题

• RQ1 在生理条件下，单体α-突触核蛋白在单分子水平上呈现哪些构象状态？
• RQ2 与疾病相关的因素（如Cu2+、A30P突变、高离子强度）如何改变α-突触核蛋白的构象平衡？
• RQ3 是否存在一种特定的α-突触核蛋白结构状态，与其中形成纤维的倾向性相关？
• RQ4 能否调节α-突触核蛋白的构象平衡以防止聚集？
• RQ5 单分子力谱能否作为筛选和验证靶向聚集通路药物的工具？

主要发现

• 在生理条件下，单体α-突触核蛋白中同时共存三种主要构象类别：无序、无规卷曲和‘β样’。
• ‘β样’构象与聚集直接相关，其在促进纤维形成的条件下显著增加。
• 在Cu2+存在下，‘β样’结构的相对丰度相比对照条件最高增加3倍。
• A30P突变增强了‘β样’构象的丰度，与其已知的致病性一致。
• 高离子强度也显著促进向‘β样’状态的转变，支持其在促进聚集中的作用。
• 本研究确立了纤维形成受化学平衡调控，且需要达到‘β样’构象的关键浓度才能触发成核。