National Centre for Biological Sciences, Tata Institute of Fundamental Research , Bangalore 560065, India.
Biochemistry. 2014 Feb 25;53(7):1206-17. doi: 10.1021/bi401467p. Epub 2014 Feb 13.
Proteins possessing very different structures, or even no structure, form amyloid fibrils that are very similar in internal structure. This suggests that the mechanisms by which amyloid fibrils form might be very similar, irrespective of whether the fibrils are associated with disease or with normal cellular function, or even if they have no physiological importance. In this context, it is important to have a model protein system whose amyloid fibril formation is robust in its reproducibility, which can reveal the fundamentals of the amyloid fibril reaction that may be applicable to all proteins. In this study, the aggregation mechanism of amyloid fibril formation by chain B of the heterodimeric protein monellin has been elucidated in detail. It is shown that the aggregation reaction meets all the stringent kinetic criteria of a homogeneous nucleation-dependent polymerization mechanism, which is valid over a wide range of protein concentrations. Quantitative analyses of the kinetic data using one approach based on features of the entire kinetic curve, and another based on only the initial rate of aggregation, indicate that the thermodynamic nucleus is a dimer. Spherical oligomers are observed by atomic force microscopy to form transiently early during fibril formation but are off-pathway to the direct fibril formation pathway. It is shown that amyloid fibril formation can be prevented by the addition of chain A of monellin at early stages of chain B aggregation: the two free chains combine to form native monellin, which leads to the dissociation of early aggregates.
具有非常不同结构甚至没有结构的蛋白质会形成非常相似的内部结构的淀粉样纤维。这表明，无论纤维是否与疾病、正常细胞功能有关，甚至是否具有生理意义，形成淀粉样纤维的机制可能非常相似。在这种情况下，拥有一个模型蛋白系统非常重要，该系统的淀粉样纤维形成具有很强的重现性，可以揭示可能适用于所有蛋白质的淀粉样纤维反应的基本原理。在这项研究中，详细阐明了杂二聚体蛋白马丁林的链 B 形成淀粉样纤维的聚集机制。结果表明，聚合反应符合均相成核依赖性聚合机制的所有严格动力学标准，该机制在广泛的蛋白质浓度范围内有效。使用一种基于整个动力学曲线特征的方法和另一种仅基于聚合初始速率的方法对动力学数据进行定量分析表明，热力学核是二聚体。原子力显微镜观察到球形寡聚物在纤维形成的早期阶段短暂形成，但不在直接纤维形成途径上。结果表明，通过在链 B 聚集的早期阶段添加马丁林的链 A 可以防止淀粉样纤维的形成：两条游离链结合形成天然马丁林，导致早期聚集体的解离。
