title: Insights into the role of the beta-2 microglobulin D-strand in amyloid propensity revealed by mass spectrometry.
abstract: In vivo beta-2 microglobulin (β2m) forms amyloid fibrils that are associated with the disease dialysis-related amyloidosis. Here, electrospray ionisation-ion mobility spectrometry-mass spectrometry has been used to compare the oligomers formed from wild-type β2m with those formed from a variant of the protein containing a single point mutation in the D strand, H51A, during in vitro fibril assembly. Using the amyloid-binding fluorescent dye, Thioflavin T, to monitor fibrillation kinetics, H51A was shown to exhibit a two-fold increase in the lag-time of fibril formation. Despite this, comparison of the oligomeric species observed during the lag-time of self-aggregation indicated that H51A had a higher population of oligomers, and formed oligomers of higher order, than wild-type β2m. The cross-sectional areas of the oligomers arising from H51A and wild-type protein were indistinguishable, although the H51A oligomers were shown to have a significantly higher kinetic stability on account of their reluctance to undergo sub-unit exchange when mixed with 15N-labelled protein. Together the data reveal a significant effect of His51, and thus that of the D-strand sequence, on amyloid formation. The results also highlight the power of mass spectrometry in probing complex biochemical mechanisms in real-time.
authors: Astbury Centre for Structural Molecular Biology and School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK. s.e.radford@leeds.ac.uk a.e.ashcroft@leeds.ac.uk.
authors: Aneika C
authors: AC
authors: Leney
authors: Clare L
authors: CL
authors: Pashley
authors: Charlotte A
authors: CA
authors: Scarff
authors: Sheena E
authors: SE
authors: Radford
authors: Alison E
authors: AE
authors: Ashcroft
mesh_terms: D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism; D000686:Amyloidosis / Q000209:etiology / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D055550:Protein Stability; D001613:beta 2-Microglobulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000682:Amyloid; D001613:beta 2-Microglobulin