Smith Andrew M, Jahn Thomas R, Ashcroft Alison E, Radford Sheena E
Astbury Centre for Structural Molecular Biology, Garstang and Astbury Buildings, University of Leeds, Leeds, UK.
J Mol Biol. 2006 Nov 17;364(1):9-19. doi: 10.1016/j.jmb.2006.08.081. Epub 2006 Sep 1.
Numerous debilitating human disorders result from protein misfolding and amyloid formation. Despite the grave nature of these maladies, our understanding of the structural mechanism of fibril assembly is limited. Of paramount importance is the need to identify and characterize oligomeric species formed early during fibril assembly, so that the nature of the initiating assembly mechanism can be revealed and species that may be toxic to cells identified. However, the transient nature of early oligomeric species, combined with their heterogeneity and instability, has precluded detailed analysis to date. Here, we have used electrospray ionisation mass spectrometry (ESI-MS), complemented by analytical ultracentrifugation (AUC) and measurements of thioflavin-T fluorescence, to monitor the early stages of assembly of amyloid-like fibrils formed from human beta-2-microglobulin (beta2m) in vitro. We show that worm-like fibrils that form with nucleation-independent kinetics assemble by a mechanism consistent with monomer addition, with species ranging from monomer to > or = 13-mer being identified directly and uniquely as transient assembly intermediates. By contrast, only monomers, dimers, trimers and tetramers are observed during nucleated growth, which leads to the formation of long straight fibrils. The results highlight the unique power of non-covalent ESI-MS to identify protein assembly intermediates in complex heterogeneous systems and demonstrate its great potential to identify and characterise individual species formed early during amyloid assembly.
许多使人衰弱的人类疾病是由蛋白质错误折叠和淀粉样蛋白形成引起的。尽管这些疾病性质严重，但我们对纤维组装结构机制的了解仍然有限。至关重要的是，需要识别和表征在纤维组装早期形成的寡聚体物种，以便揭示起始组装机制的本质，并识别可能对细胞有毒的物种。然而，早期寡聚体物种的瞬态性质，加上它们的异质性和不稳定性，迄今为止排除了详细分析。在这里，我们使用电喷雾电离质谱（ESI-MS），辅以分析超速离心（AUC）和硫黄素-T荧光测量，来监测体外由人β-2-微球蛋白（β2m）形成的淀粉样样纤维组装的早期阶段。我们表明，以非成核动力学形成的蠕虫状纤维通过与单体添加一致的机制组装，直接且独特地鉴定出从单体到≥13聚体的物种作为瞬态组装中间体。相比之下，在成核生长过程中仅观察到单体、二聚体、三聚体和四聚体，这导致形成长直纤维。结果突出了非共价ESI-MS在识别复杂异质系统中蛋白质组装中间体方面的独特能力，并证明了其在识别和表征淀粉样蛋白组装早期形成的单个物种方面的巨大潜力。
