Partially Unfolded States of β2-Microglobulin and Amyloid Formation in Vitro

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Abstract
Dialysis-related amyloidosis (DRA) involves the aggregation of β2-microglobulin (β2m) into amyloid fibrils. Using Congo red and thioflavin-T binding, electron microscopy, and X-ray fiber diffraction, we have determined conditions under which recombinant monomeric β2m spontaneously associates to form fibrils in vitro. Fibrillogenesis is critically dependent on the pH and the ionic strength of the solution, with low pH and high ionic strength favoring fibril formation. The morphology of the fibrils formed varies with the growth conditions. At pH 4 in 0.4 M NaCl the fibrils are ∼10 nm wide, relatively short (50−200 nm), and curvilinear. By contrast, at pH 1.6 the fibrils formed have the same width and morphology as those formed at pH 4 but extend to more than 600 nm in length. The dependence of fibril growth on ionic strength has allowed the conformational properties of monomeric β2m to be determined under conditions where fibril growth is impaired. Circular dichroism studies show that titration of one or more residues with a pKa of 4.7 destabilizes native β2m and generates a partially unfolded species. On average, these molecules retain significant secondary structure and have residual, non-native tertiary structure. They also bind the hydrophobic dye 1-anilinonaphthalene-8-sulfonic acid (ANS), show line broadening in one-dimensional 1H NMR spectra, and are weakly protected from hydrogen exchange. Further acidification destabilizes this species, generating a second, more highly denatured state that is less fibrillogenic. These data are consistent with a model for β2m fibrillogenesis in vitro involving the association of partially unfolded molecules into ordered fibrillar assemblies.