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Investigating the structural properties of amyloid‐like fibrils formed in vitro from β 2 ‐microglobulin using limited proteolysis and electrospray ionisation mass spectrometry
Author(s) -
Myers Sarah L.,
Thomson Neil H.,
Radford Sheena E.,
Ashcroft Alison E.
Publication year - 2006
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.2482
Subject(s) - fibril , chemistry , proteolysis , ionic strength , biophysics , mass spectrometry , peptide , amyloid (mycology) , crystallography , biochemistry , chromatography , enzyme , organic chemistry , aqueous solution , inorganic chemistry , biology
The protein β 2 ‐microglobulin ( β 2 m) aggregates to form classical amyloid fibrils in patients undergoing long‐term haemodialysis. Amyloid‐like fibrils with a cross‐ β fold can also be formed from wild‐type β 2 m under acidic conditions in vitro . The morphology of such fibrils depends critically on the conditions used: incubation of β 2 m in low ionic strength buffers at pH 2.5 results in the formation of long (µm), 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 β 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 β 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 β 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‐ β architecture. Copyright © 2006 John Wiley & Sons, Ltd.