Understanding the amyloid fibril formation of β2-microglobulin on the basis of protein conformation

β-2-微球蛋白(β-2-m)相关性淀粉样变性是长期血液透析患者的严重并发症。为了了解β2-m形成淀粉原纤维的机制，我们一直在研究重组人β2-m的构象和淀粉原纤维的形成。1.我们建立了一种新的方法，利用酰胺质子的H/D交换结合核磁共振分析来表征单残基分辨率下β2-m淀粉原纤维的构象柔性。结果表明，分子中间区域的大部分残基，包括天然结构中的环区，形成了一个刚性的β-Sheet核心，而N-端和C-端不是这个核心的一部分。交换时间曲线偏离了单一指数曲线，与纤维的超分子结构相一致。2.另一方面，对纤维生长的实时监测对于阐明纤维形成的机理是必不可少的。硫黄素T(THT)是一种已知的试剂，在与淀粉样蛋白纤维结合时会变得强烈荧光。我们发现，通过用全内反射荧光显微镜监测β2-m的荧光，可以在不需要共价荧光标记的情况下观察到淀粉样纤维。该方法用于跟踪种子依赖的β2-m原纤维伸展动力学，揭示了单向伸展。由于THT结合对淀粉样蛋白纤维是常见的，这种方法将具有普遍的适用性，正如阿尔茨海默氏症淀粉样蛋白β肽所证实的那样。

β2-Microglobulin (β2-m)-related amyloidosis is a serious complication in patients receiving long-term hemodialysis. To understand the mechanism of amyloid fibril formation by β2-m, we have been studying the conformation and amyloid fibril formation of recombinant human β2-m.1.We established a novel procedure using H/D exchange of amide protons combined with NMR analysis for characterizing the conformational flexibility of β2-m amyloid fibrils at single-residue resolution. The results indicated that most residues in the middle region of the molecule, including the loop regions in the native structure, form a rigid β-sheet core, while the N-and C-termini are not part of this core. The exchange time course deviated largely from a single exponential curve, consistent with the supramolecular structure of fibrils.2.On the other hand, real-time monitoring of fibril growth is essential to clarify the mechanism of fibril formation. Thioflavin T (ThT) is a reagent known to become strongly fluorescent upon binding to amyloid fibrils. We show that, by monitoring ThT fluorescence with total internal reflection fluorescence microscopy, amyloid fibrils of β2-m can be visualized without requiring covalent fluorescence labeling. This method was used to follow the kinetics of seed-dependent β2-m fibril extension, revealing the unidirectional extension. Since ThT binding is common to amyloid fibrils, this method will have general applicability as confirmed with the Alzheimer's amyloid β-peptide.