Dataset Information

Elongated oligomers in beta2-microglobulin amyloid assembly revealed by ion mobility spectrometry-mass spectrometry.

ABSTRACT: The key to understanding amyloid disease is the characterization of oligomeric species formed during the early stages of fibril assembly. Here we have used electrospray ionisation-ion mobility spectrometry-mass spectrometry to identify and structurally characterize the oligomers formed during amyloid assembly from beta(2)-microglobulin (beta(2)m). Beta(2)m oligomers are shown to have collision cross-sections consistent with monomeric units arranged in elongated assemblies prior to fibril formation. Direct observation, separation, and quantification of transient oligomeric species reveals that monomers to tetramers are populated through the lag phase with no evidence for the significant population of larger oligomeric species under the conditions employed. The dynamics of each oligomeric species were monitored directly within the ensemble at concentrations commensurate with amyloid formation by observing the subunit exchange of (14)N- and (15)N-labeled oligomers. Analysis of the data revealed a decrease in oligomer dynamics concomitant with increasing oligomer size and the copopulation of dynamic dimeric and trimeric species with more stable trimeric and tetrameric species. The results presented map the events occurring during the lag phase of fibril formation and give a clear insight into the structural characteristics and dynamic nature of the beta(2)m oligomers, demonstrating the existence of elongated assemblies arising from an intact amyloidogenic protein during fibril formation.

SUBMITTER: Smith DP

PROVIDER: S-EPMC2872402 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Elongated oligomers in beta2-microglobulin amyloid assembly revealed by ion mobility spectrometry-mass spectrometry.

Smith David P DP Radford Sheena E SE Ashcroft Alison E AE

Proceedings of the National Academy of Sciences of the United States of America 20100329 15

The key to understanding amyloid disease is the characterization of oligomeric species formed during the early stages of fibril assembly. Here we have used electrospray ionisation-ion mobility spectrometry-mass spectrometry to identify and structurally characterize the oligomers formed during amyloid assembly from beta(2)-microglobulin (beta(2)m). Beta(2)m oligomers are shown to have collision cross-sections consistent with monomeric units arranged in elongated assemblies prior to fibril formati ...[more]

PMID: 20351246

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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

Elongated oligomers in beta2-microglobulin amyloid assembly revealed by ion mobility spectrometry-mass spectrometry.

Publications

Elongated oligomers in beta2-microglobulin amyloid assembly revealed by ion mobility spectrometry-mass spectrometry.

Similar Datasets

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