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

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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.