Human stefin B readily forms amyloid fibrils in vitro

Limited Proteolysis Reveals That Amyloids from the 3D Domain-Swapping Cystatin B Have a Non-Native β-Sheet Topology

“…These conditions produce mature fibres in a suitable experimental timescale of 1 week and are the most studied to date [reviewed in 22,35]. Cystatin B fibrils produced in these conditions are 3.4 (±0.3) nm in height by AFM [19], whilst single filaments are 8.6 (SD±1.4) nm wide and tens of micrometers in length in TEM images (Fig. 2c).…”

“…Amyloid fibrils were formed by 37°C incubation of 30 µM cystatin B in pH 4.7, 15 mM sodium acetate, 100 mM NaCl, 1 mM sodium azide and 10% (v/v) 2,2,2-trifluoroethanol[19]. Soluble cystatin B preparations and amyloid fibrils are entirely composed of full length 1-98 residue protein, determined by MS. Fibril morphology was examined by TEM following a minimum three week incubation which leads to 100% soluble to insoluble protein conversion and almost exclusive formation of amyloid fibrils as determined by TEM and subsequent UV spectroscopy of soluble fractions following centrifugation.…”

Essential Role of Proline Isomerization in Stefin B Tetramer Formation

“…[5][6][7][8][9] Protein self-association has been shown to involve several kinds of intermediate species and can result in different kinds of amorphous and fibrilar aggregates. 10 During the lagphase that normally precedes fibril formation, dimers 8,11 and higher oligomers may accumulate prior to the exponential growth phase. 12 One of the models describing how amyloid fibril formation can occur from globular proteins involves 3D domainswapping, [13][14][15] where the basic building units of amyloid fibrils may be domain-swapped dimers or endless chain of so called run away domainswapped subunits.…”

Structure and Function of Stefin B Oligomers – Important Role in Amyloidogenesis

“…That stefin B forms amyloid fibrils in vitro under relatively mild conditions was first shown in 2002 [58] and various solution conditions were probed, fibrils and protofibrils were imaged by AFM and TEM [59]. The fibril formation of stefin B was compared to that of stefin A, a much more stable homologue [60], to proline mutants [61], EPM1 mutants [62] and to chimeras between stefins A and B [63].…”