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Partially unfolded states of beta(2)-microglobulin and amyloid formation in vitro

McParland, V J, Kad, Neil M, Kalverda, A P, Brown, A, Kirwin-Jones, P, Hunter, M G, Sunde, M, Radford, S E (2000) Partially unfolded states of beta(2)-microglobulin and amyloid formation in vitro. Biochemistry, 39 (30). pp. 8735-8746. ISSN 0006-2960. (doi:10.1021/bi000276j) (The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided) (KAR id:42954)

The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided. (Contact us about this Publication)
Official URL
http://dx.doi.org/10.1021/bi000276j

Abstract

Dialysis-related amyloidosis (DRA) involves the aggregation of beta(2)-microglobulin (beta(2)m) 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 beta(2)m 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 approximately 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 beta(2)m to be determined under conditions where fibril growth is impaired. Circular dichroism studies show that titration of one or more residues with a pK(a) of 4.7 destabilizes native beta(2)m 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 (1)H 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 beta(2)m fibrillogenesis in vitro involving the association of partially unfolded molecules into ordered fibrillar assemblies.

Item Type: Article
DOI/Identification number: 10.1021/bi000276j
Subjects: Q Science
Divisions: Faculties > Sciences > School of Biosciences
Depositing User: Neil Kad
Date Deposited: 15 Sep 2014 19:08 UTC
Last Modified: 06 May 2020 03:10 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/42954 (The current URI for this page, for reference purposes)
Kad, Neil M: https://orcid.org/0000-0002-3491-8595
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