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Mapping the Core of the B2-Microglobulin Amyloid Fibril by H/D Exchange




Amyloid fibril deposition is associated with 20 serious human diseases including Alzheimer’s disease, transmissible spongiform encephalopathies and dialysis-related amyloidosis (1, 2). The amyloid hypothesis identifies the amyloid fibrils as the cause of these diseases. Electron microscopy revealed that most of the amyloid fibrils assume a long, unbranched and straight fibrous structure with a uniform diameter of ~10 nm and of longitudinal periodicity. The results of X-ray fiber diffraction analysis suggested that amyloidgenic precursor proteins assemble into a cross-ß-fiber structure. However, amyloid fibrils are non-crystalline assemblies of high molecular weight. This precludes the use of conventional methods such as X-ray crystallography or multidimensional NMR to acquire high resolution structural images. Thus, even though the genetic evidence in favor of amyloid hypothesis is compelling, the lack of detailed structural information has retarded clarification of the mechanism of amyloid fibril formation and their role in amyloidosis. Here, we describe a novel procedure using 1H/2H (H/D) exchange of amide protons combined with NMR analysis for characterizing the conformational flexibility of ß2-microglobulin ( ß2-m) amyloid fibrils at single-residue resolution.

Dialysis-related amyloidosis

In the Japan alone, more than 210,000 people have impaired renal function that requires treatment by hemodialysis. ß2-m amyloidosis (or dialysis-related amyloidosis) is a common and serious complication in patients receiving hemodialysis for more than 10 years (Fig. 1) (3). The native ß2-m, a 99-residue protein with a typical immunoglobulin fold, exists on the surface of nearly all cells as part of the light chain of the major histocompatibility complex class 1 antigen (4) and is a normal constituent of plasma (Fig. 2). The daily production of ß2-m is in the range of 150 - 200 mg/day, of which 97% is excreted in the kidneys. The concentration of ß2-m, therefore, is significantly increased in patients with renal failure. More seriously, hemodialysis therapy results in the retention of a high concentration of ß2-m in the plasma. Although it is evident that the elevated concentration of ß2-m in plasma for a long-term causes amyloidosis, the molecular mechanism of amyloid fibril formation is unclear. In contrast, in vitro, needle-like amyloid fibrils are formed by the seed-dependent extension reaction at pH 2.5, in which monomeric ß2-m is added to the seed fibrils (Fig. 2) (5).


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