Different propensity to form amyloid fibrils by two homologous proteins-Human stefins A and B: searching for an explanation.
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 A and 10 A reflections characteristic for cross beta-structure. Similar height of approximately 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 microscopy (TEM), was in the same range as previously reported for stefin B (Zerovnik 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 alpha-helix (and the alpha-loop) to beta-sheet in stefin A monomeric and dimeric forms.
Jenko
Sasa
S
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Ljubljana, Slovenia.
Skarabot
Miha
M
Kenig
Manca
M
Guncar
Gregor
G
Musevic
Igor
I
Turk
Dusan
D
Zerovnik
Eva
E
D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D055313:Cystatin B; D015891:Cystatins / Q000737:chemistry* / Q000378:metabolism*; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D008280:Magnetics; D018625:Microscopy, Atomic Force; D008854:Microscopy, Electron; D008958:Models, Molecular; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D012997:Solvents / Q000737:chemistry; D013329:Structure-Activity Relationship
D000682:Amyloid; C524994:CSTB protein, human; D015891:Cystatins; D012997:Solvents; D055313:Cystatin B