Investigating the structural properties of amyloid-like fibrils formed in vitro from beta2-microglobulin using limited proteolysis and electrospray ionisation mass spectrometry.
The protein beta(2)-microglobulin (beta(2)m) aggregates to form classical amyloid fibrils in patients undergoing long-term haemodialysis. Amyloid-like fibrils with a cross-beta fold can also be formed from wild-type beta(2)m under acidic conditions in vitro. The morphology of such fibrils depends critically on the conditions used: incubation of beta(2)m in low ionic strength buffers at pH 2.5 results in the formation of long (microm), straight fibrils while, at pH 3.6, short (<500 nm) fibrils form. At higher ionic strengths (0.2-0.4 M) at pH 1.5-3.6, the fibrils have a distinct curved and nodular morphology. To determine the conformational properties of beta(2)m within in vitro fibrils of different morphologies, limited proteolysis of each fibril type using pepsin was performed and the resulting peptide fragments identified by tandem mass spectrometry. For comparison, the proteolytic degradation patterns of monomeric beta(2)m and seven synthetic peptides spanning the entire sequence of the intact protein were similarly analysed. The results show that fibrils with different morphologies result in distinct digestion patterns. While the curved, worm-like fibrils are relatively weakly protected from proteolysis, the long, straight fibrils formed at pH 2.5 at low ionic strength show only a single cut-site at Val9, demonstrating that substantial refolding of the initially acid-denatured and unprotected state of beta(2)m occurs during assembly. The data demonstrate that the organisation of the polypeptide chain in fibrils with different morphological features differs considerably, despite the fact that the fibrils possess a common cross-beta architecture.
Myers
Sarah L
SL
Astbury Centre for Structural Molecular Biology, Astbury & Garstang Buildings, University of Leeds, Leeds LS2 9JT, UK.
Thomson
Neil H
NH
Radford
Sheena E
SE
Ashcroft
Alison E
AE
D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry* / Q000648:ultrastructure; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D018625:Microscopy, Atomic Force; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D001613:beta 2-Microglobulin / Q000737:chemistry*
D000682:Amyloid; D001613:beta 2-Microglobulin; D010434:Pepsin A