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Insights into the role of the beta-2 microglobulin D-strand in amyloid propensity revealed by mass spectrometry.

ABSTRACT: In vivo beta-2 microglobulin (?2m) 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 ?2m 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 ?2m. 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.

SUBMITTER: Leney AC

PROVIDER: S-EPMC4006425 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Insights into the role of the beta-2 microglobulin D-strand in amyloid propensity revealed by mass spectrometry.

Leney Aneika C AC Pashley Clare L CL Scarff Charlotte A CA Radford Sheena E SE Ashcroft Alison E AE

Molecular bioSystems 20131212 3

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 wa ...[more]

PMID: 24336936

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Project description:Beta-2-microglobulin (beta2m) deposits as amyloid fibrils in the musculoskeletal system of patients undergoing long-term dialysis treatment as a result of kidney failure. Previous work has shown that Cu(II) binding causes beta2m to organize into nativelike dimers and tetramers that precede amyloid formation. Cu(II) is then released from higher-order oligomers before mature Cu(II)-free amyloid fibrils are formed. While some of the Cu(II)-induced structural changes that enable beta2m self-assembly are starting to be revealed, the details of how the Cu(II) binding site evolves from the monomer to the dimers and tetramers are not known. Here, we report results from three mass spectrometry (MS)-based methods that provide insight into the changing Cu-beta2m interactions. We find that monomeric beta2m binds Cu(II) via the N-terminal amine, the amide of Gln2, His31, and Asp59. In the dimer and tetramer, Asp59 is no longer bound to Cu(II), but the other residues still comprise a well-defined albeit weaker binding site that is better able to release Cu(II). Consistent with this is the observation that a fraction of the tetrameric species no longer binds Cu(II) at this weakened binding site, which agrees with a previous report that suggested the tetramer as the first Cu(II)-free oligomer. Our results also provide some insight into structural changes caused by Cu(II) binding that facilitate oligomer formation. Specifically, binding by Asp59 in the monomer requires significant movement of this residue, and we propose that this repositioning is important for establishing a pair of dimer-stabilizing salt bridges between this residue and Lys19. We also find evidence that Cu(II) binding in the N-terminal region of the monomer repels Arg3, which likely allows this residue to form a pair of dimer-stabilizing salt bridges with Glu16. Overall, our measurements suggest that the previously proposed conformational switch caused by Cu(II) binding includes not only a cis-trans isomerization at Pro32 but also the repositioning of residues that are critical for the formation of new electrostatic interactions.

Dataset Information

Insights into the role of the beta-2 microglobulin D-strand in amyloid propensity revealed by mass spectrometry.

Publications

Insights into the role of the beta-2 microglobulin D-strand in amyloid propensity revealed by mass spectrometry.

Similar Datasets

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