Fujiwara Satoru, Matsumoto Fumiko, Yonezawa Yasushige
Neutron Science Research Center, Japan Atomic Energy Research Institute, Tokai-mura, Naka-gun, 319-1195, Ibaraki, Japan.
J Mol Biol. 2003 Aug 1;331(1):21-8. doi: 10.1016/s0022-2836(03)00722-8.
Various proteins have been shown to form various aggregated structures including the filamentous aggregates known as amyloid fibrils depending on the solution conditions. Hen egg white lysozyme (HEWL) is one of the proteins that form the amyloid fibrils. To gain insight into the mechanism of this polymorphism of the aggregated structures, we employed a model system consisting of HEWL, pure water, and ethanol, and investigated the kinetic process of the fibril formation in various salt concentrations with time-resolved neutron scattering. It was shown that by addition of NaCl in a range between 0.3 mM and 1.0 mM to HEWL solution in 90% ethanol, gelation occurred, and this gelation proceeded through a two-step process: the lateral association of the protofilaments, followed by the cross-linking of these fibrils formed. Both the structures of the fibrils and the rate of the gelation depended on NaCl concentration. The average structures of the fibrils formed at 1.0 mM NaCl were characterized by the radius of gyration of their cross-section (45.9(+/-0.4)A) and the number of the protofilaments within the fibril (4.10(+/-0.12)), corresponding to the mature amyloid fibrils. A range of intermediate structures was formed below 1 mM NaCl. Above 2 mM NaCl, precipitation occurred because of the formation of amorphous aggregates. Here the branch point to the formation of the mature amyloid fibrils or to the amorphous aggregates was after the formation of the protofilaments. Sensitivity of the aggregated structures to salt concentration suggests that electrostatic interaction plays an essential role in the formation of these structures. The structural diversity both in the fibrils and the aggregated structures of the fibrils can be interpreted in terms of the difference in the degree of the electrostatic shielding at different salt concentrations.
根据溶液条件，各种蛋白质已被证明能形成各种聚集结构，包括被称为淀粉样纤维的丝状聚集体。鸡蛋清溶菌酶（HEWL）是形成淀粉样纤维的蛋白质之一。为了深入了解这种聚集结构多态性的机制，我们采用了一个由HEWL、纯水和乙醇组成的模型系统，并通过时间分辨中子散射研究了在各种盐浓度下纤维形成的动力学过程。结果表明，在90%乙醇中的HEWL溶液中加入0.3 mM至1.0 mM范围内的NaCl时，会发生凝胶化，且这种凝胶化过程分两步进行：原纤维的横向缔合，随后是这些形成的纤维的交联。纤维的结构和凝胶化速率均取决于NaCl浓度。在1.0 mM NaCl下形成的纤维的平均结构通过其横截面的回转半径（45.9（±0.4）Å）和纤维内原纤维的数量（4.10（±0.12））来表征，这与成熟的淀粉样纤维相对应。在1 mM NaCl以下形成了一系列中间结构。在2 mM NaCl以上，由于无定形聚集体的形成而发生沉淀。这里，形成成熟淀粉样纤维或无定形聚集体的分支点是在原纤维形成之后。聚集结构对盐浓度的敏感性表明静电相互作用在这些结构的形成中起着至关重要的作用。纤维及其聚集结构中的结构多样性可以用不同盐浓度下静电屏蔽程度的差异来解释。
