Different propensity to form amyloid fibrils by two homologous proteins—Human stefins A and B: Searching for an explanation

Abstract

By using ThT fluorescence, X-ray diffraction, and atomic force microscopy (AFM), it has been shown that human stefins A and B (subfamily A of cystatins) form amyloid fibrils. Both protein fibrils show the 4.7 Å and 10 Å reflections characteristic for cross β-structure. Similar height of ∼3 nm and longitudinal repeat of 25–27 nm were observed by AFM for both protein fibrils. Fibrils with a double height of 5.6 nm were only observed with stefin A. The fibril's width for stefin A fibrils, as observed by transmission electron microsopy (TEM), was in the same range as previously reported for stefin B (Žerovnik et al., Biochem Biophys Acta 2002;1594:1–5). The conditions needed to undergo fibrillation differ, though. The amyloid fibrils start to form at pH 5 for stefin B, whereas in stefin A, preheated sample has to be acidified to pH < 2.5. In both cases, adding TFE, seeding, and alignment in a strong magnetic field accelerate the fibril growth. Visual analysis of the three-dimensional structures of monomers and domain-swapped dimers suggests that major differences in stability of both homologues stem from arrangement of specific salt bridges, which fix α-helix (and the α-loop) to β-sheet in stefin A monomeric and dimeric forms. Proteins 2004;55:000–000.