National Centre for Biological Sciences, Tata Institute of Fundamental Research , Bangalore 560065, India.
Biochemistry. 2014 Oct 21;53(41):6550-65. doi: 10.1021/bi500528x. Epub 2014 Oct 6.
The aggregation of the microtubule-associated protein, tau, into amyloid fibrils is a hallmark of neurodegenerative diseases such as the tauopathies and Alzheimer's disease. Since monomeric tau is an intrinsically disordered protein and the polymeric fibrils possess an ordered cross-β core, the aggregation process is known to involve substantial conformational conversion besides growth. The aggregation mechanism of tau in the presence of inducers such as heparin, deciphered using probes such as thioflavin T/S fluorescence, light scattering, and electron microscopy assays, has been shown to conform to ligand-induced nucleation-dependent polymerization. These probes do not, however, distinguish between the processes of conformational conversion and fibril growth. In this study, UV resonance Raman spectroscopy is employed to look directly at signatures of changes in secondary structure and side-chain packing during fibril formation by the four repeat functional domain of tau in the presence of the inducer heparin, at pH 7 and at 37 °C. Changes in the positions and intensities of the amide Raman bands are shown to occur in two distinct stages during the fibril formation process. The first stage of UVRR spectral changes corresponds to the transformation of monomer into early fibrillar aggregates. The second stage corresponds to the transformation of these early fibrillar aggregates into the final, ordered, mature fibrils and during this stage; the processes of conformational conversion and the consolidation of the fibril core occur simultaneously. Delineation of these secondary structural changes accompanying the formation of tau fibrils holds significance for the understanding of generic and tau-specific principles of amyloid assembly.
微管相关蛋白 tau 的聚集形成淀粉样纤维是神经退行性疾病（如 tau 病和阿尔茨海默病）的一个标志。由于单体 tau 是一种固有无序的蛋白质，而聚合纤维具有有序的交叉-β核心，因此聚合过程除了生长外还涉及到大量的构象转换。使用硫黄素 T/S 荧光、光散射和电子显微镜等探针，已经揭示了在肝素等诱导剂存在下 tau 的聚合机制，符合配体诱导的成核依赖性聚合。然而，这些探针并不能区分构象转换和纤维生长的过程。在这项研究中，我们采用紫外共振拉曼光谱直接观察在诱导剂肝素存在下，四重复功能域 tau 在 pH 7 和 37°C 下形成纤维时二级结构和侧链堆积的变化特征。在纤维形成过程中，酰胺拉曼带的位置和强度的变化被证明发生在两个不同的阶段。UVRR 光谱变化的第一阶段对应于单体向早期纤维状聚集体的转化。第二阶段对应于这些早期纤维状聚集体向最终有序成熟纤维的转化，在此阶段，构象转换和纤维核心的巩固同时发生。这些伴随着 tau 纤维形成的二级结构变化的描述对于理解通用和 tau 特异性淀粉样蛋白组装原则具有重要意义。
