1 Risø National Laboratory for Sustainable Energy, Technical University of Denmark2 unknown3 Department of Micro- and Nanotechnology, Technical University of Denmark
beta(2)-Microglobulin (beta(2)m) is the amyloidogenic protein in dialysis-related amyloidosis, but the mechanisms underlying beta(2)m fibrillogenesis in vivo are largely unknown. We study a structural variant of beta(2)M that has been linked to cancer and inflammation and may be present in the circulation of dialysis patients. This beta(2)M variant, Delta K58-beta(2)m, is a disulfide-linked two-chain molecule consisting of amino acid residues 1-57 and 59-99 of intact beta(2)m, and we here demonstrate and characterize its decreased conformational stability as compared to wild-type (wt) beta(2)M. Using amide hydrogen/deuterium exchange monitored by mass spectrometry, we show that Delta K58-beta(2)m has increased unfolding rates compared to wt-beta(2)m and that unfolding is highly temperature dependent. The unfolding rate is I order of magnitude faster in Delta K58-beta(2)M than in wt-beta(2)m, and at 37 degrees C the half-time for unfolding is more than 170-fold faster than at 15 degrees C. Conformational changes are also reflected by a very prominent Congo red binding of Delta K58-beta(2)m at 37 degrees C, by the evolution of thioflavin T fluorescence, and by changes in intrinsic fluorescence. After a few days at 37 degrees C, in contrast to wt-beta(2)M, Delta K-58-beta(2)M forms well-defined high molecular weight aggregates that are detected by size-exclusion chromatography. Atomic force microscopy after seeding with amyloid-beta(2)m fibrils under conditions that induce minimal fibrillation in wt-beta(2)m shows extensive amyloid fibrillation in Delta K58-beta(2)m samples. The results highlight the instability and amyloidogenicity under near physiological conditions of a slightly modified beta(2)m variant generated by limited proteolysis and illustrate stages of amyloid formation from early conformational variants to overt fibrillation.
Biochemistry, 2005, Vol 44, Issue 11, p. 4397-4407