Kad Neil M, Myers Sarah L, Smith David P, Smith D Alastair, Radford Sheena E, Thomson Neil H
School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
J Mol Biol. 2003 Jul 18;330(4):785-97. doi: 10.1016/s0022-2836(03)00583-7.
The kinetics of spontaneous assembly of amyloid fibrils of wild-type beta(2)-microglobulin (beta(2)M) in vitro, under acid conditions (pH 2.5) and low ionic strength, has been followed using thioflavin-T (ThT) binding. In parallel experiments, the morphology of the different fibrillar species present at different time-points during the growth process were characterised using tapping-mode atomic force microscopy (TM-AFM) in air and negative stain electron microscopy (EM). The thioflavin-T assay shows a characteristic lag phase during which the nucleation of fibrils occurs before a rapid growth in fibril density. The volume of fibrils deposited on mica measured from TM-AFM images at each time-point correlates well with the fluorescence data. TM-AFM and negative-stain EM revealed the presence of various kinds of protein aggregates in the lag phase that disappear concomitantly with a rise in the density of amyloid fibrils, suggesting that these aggregates precede fibril growth and may act as nucleation sites. Three distinct morphologies of mature amyloid fibrils were observed within a single growth experiment, as observed previously for the wild-type protein and the variant N17D. Additional supercoiled morphologies of the lower-order fibrils were observed. Comparative height analysis from the TM-AFM data allows each of the mature fibril types and single protofilaments to be identified unambiguously, and reveals that the assembly occurs via a hierarchy of morphological states.
在酸性条件（pH 2.5）和低离子强度下，利用硫黄素-T（ThT）结合追踪了野生型β2-微球蛋白（β2M）淀粉样纤维在体外的自发组装动力学。在平行实验中，使用空气中的轻敲模式原子力显微镜（TM-AFM）和负染电子显微镜（EM）对生长过程中不同时间点存在的不同纤维种类的形态进行了表征。硫黄素-T检测显示出一个特征性的延迟期，在此期间纤维的成核发生在纤维密度快速增长之前。从每个时间点的TM-AFM图像测量的沉积在云母上的纤维体积与荧光数据相关性良好。TM-AFM和负染EM揭示了在延迟期存在各种蛋白质聚集体，它们随着淀粉样纤维密度的增加而同时消失，这表明这些聚集体先于纤维生长，可能作为成核位点。在单个生长实验中观察到了成熟淀粉样纤维的三种不同形态，这与之前对野生型蛋白和变体N17D的观察结果一致。还观察到了低阶纤维的额外超螺旋形态。从TM-AFM数据进行的比较高度分析能够明确识别每种成熟纤维类型和单个原纤维，并揭示组装是通过形态状态的层次结构发生的。
