Despite numerous efforts, the lack of detailed structural information on amyloid fibrils has hindered clarification of the mechanism of their formation. Here, we describe a novel procedure for characterizing the conformational flexibility of beta(2)-microglobulin amyloid fibrils at single-residue resolution that uses H/D exchange of amide protons combined with NMR analysis. The results indicate that most residues in the middle region of the molecule, including the loop regions in the native structure, form a rigid beta-sheet core, whereas the the N- and C-termini are excluded from this core. The extensively hydrogen-bonded beta-sheet core explains the remarkable rigidity and stability of amyloid fibrils. The present method could be used to obtain residue-specific conformational information of various amyloid fibrils, even though it does not provide a high resolution three-dimensional structure.

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To obtain insight into the mechanism of amyloid fibril formation from ␤ 2 -microglobulin (␤2-m), we prepared a series of peptide fragments using a lysine-specific protease from Achromobacter lyticus and examined their ability to form amyloid fibrils at pH 2.5. Among the nine peptides prepared by the digestion, the peptide Ser 20 -Lys 41 (K3) spontaneously formed amyloid fibrils, confirmed by thioflavin T binding and electron microscopy. The fibrils composed of K3 peptide induced fibril formation of intact ␤2-m with a lag phase, distinct from the extension reaction without a lag phase observed for intact ␤2-m seeds. Fibril formation of K3 peptide with intact ␤2-m seeds also exhibited a lag phase. On the other hand, the extension reaction of K3 peptide with the K3 seeds occurred without a lag phase. At neutral pH, the fibrils composed of either intact ␤2-m or K3 peptide spontaneously depolymerized. Intriguingly, the depolymerization of K3 fibrils was faster than that of intact ␤2-m fibrils. These results indicated that, although K3 peptide can form fibrils by itself more readily than intact ␤2-m, the K3 fibrils are less stable than the intact ␤2-m fibrils, suggesting a close relation between the free energy barrier of amyloid fibril formation and its stability.Many proteins and peptides form amyloid fibrils (1-3). Although most are related to diseases, it has been shown that several proteins (4, 5) and peptides (6, 7) that are not related to disease can also form amyloid fibrils. Amyloid fibril formation is now recognized as a phenomenon common to many proteins. On the other hand, amyloid fibrils are homogeneous, and it is rarely possible to form chimeric fibrils composed of distinct amyloid proteins or peptides (8,9). This high species barrier suggests that the amyloid fibrils are stabilized by specific interactions of amyloid proteins, which are governed by the characteristic primary and higher order structures of each amyloid protein. Thus, amyloid fibrils can be considered to be alternatively folded conformations of globular proteins, and understanding of their properties is essential to obtain further insight into the mechanism of protein folding.␤ 2 -Microglobulin (␤2-m) 1 -related amyloidosis is a common and serious complication in patients on long term hemodialysis (10 -12). Carpal tunnel syndrome and destructive arthropathy associated with cystic bone lesions are the major clinical manifestations of ␤2-m-related amyloidosis (13). Although ␤2-m, the light chain of the type I major histocompatibility complex (14, 15), was identified as a major structural component of amyloid fibrils deposited in the synovia of the carpal tunnel (10), the mechanism of amyloid fibril formation by ␤2-m is still unknown (16,17,25,26). Naiki and co-workers (18 -20) have been studying the amyloid fibril formation of ␤2-m as well as other amyloid fibrils (21-24). They established a kinetic experimental system to analyze amyloid fibril formation in vitro, in which the extension phase with the seed fibrils is quantitatively characte...