Designing conditions for in vitro formation of amyloid protofilaments and fibrils
Designing conditions for in vitro formation of amyloid protofilaments and fibrils
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DOI：
10.1073/pnas.96.7.3590
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发表时间：
1999-03-30
影响因子：
11.1
通讯作者：
Dobson, CM
中科院分区：
文献类型：
--
作者：
Chiti, F;Webster, P;Dobson, CM
我们已经能够将一种小的α/β蛋白，酰基磷酸酶，从其可溶性和天然形式转化为在一系列病理条件下观察到的类型的不溶性淀粉样蛋白原纤维。这是通过在含有中等浓度三氟乙醇的溶液中缓慢生长来实现的。当用电子显微镜分析时，长时间孵育后样品中存在的蛋白质聚集体由30-50埃宽的延伸的无分支细丝组成，其随后组装成更高级的结构。这种纤维状材料具有广泛的β-折叠结构，如远紫外CD和IR光谱所揭示的。此外，原纤维表现出刚果红双折射，硫代黄素T的荧光增加，并导致刚果红吸收光谱的红移。所有这些特征都是淀粉样纤维的典型特征。结果表明，当蛋白质的天然折叠在多肽链内的非共价相互作用（特别是氢键）保持有利的条件下不稳定时，淀粉样蛋白的形成发生。我们认为，淀粉样蛋白的形成并不局限于少数蛋白质序列，而是在适当条件下许多（如果不是全部）天然多肽链的共同特性。
We have been able to convert a small alpha/beta protein, acylphosphatase, from its soluble and native form into insoluble amyloid fibrils of the type observed in a range of pathological conditions. This was achieved by allowing slow growth in a solution containing moderate concentrations of trifluoroethanol. When analyzed with electron microscopy, the protein aggregate present in the sample after long incubation times consisted of extended, unbranched filaments of 30-50 Angstrom in width that assemble subsequently into higher order structures. This fibrillar material possesses extensive beta-sheet structure as revealed by far-UV CD and IR spectroscopy. Furthermore, the fibrils exhibit Congo red birefringence, increased fluorescence with thioflavine T and cause a redshift of the Congo red absorption spectrum. All of these characteristics are typical of amyloid fibrils. The results indicate that formation of amyloid occurs when the native fold of a protein is destabilized under conditions in which noncovalent interactions, and in particular hydrogen bonding, within the polypeptide chain remain favorable. We suggest that amyloid formation is not restricted to a small number of protein sequences but is a property common to many, if not all, natural polypeptide chains under appropriate conditions.