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Amyloid formation under physiological conditions proceeds via a native-like folding intermediate

Nature Structural & Molecular Biology volume 13, pages 195–201 (2006)Cite this article

Abstract

Although most proteins can assemble into amyloid-like fibrils in vitro under extreme conditions, how proteins form amyloid fibrils in vivo remains unresolved. Identifying rare aggregation-prone species under physiologically relevant conditions and defining their structural properties is therefore an important challenge. By solving the folding mechanism of the naturally amyloidogenic protein β-2-microglobulin at pH 7.0 and 37 °C and correlating the concentrations of different species with the rate of fibril elongation, we identify a specific folding intermediate, containing a non-native trans-proline isomer, as the direct precursor of fibril elongation. Structural analysis using NMR shows that this species is highly native-like but contains perturbation of the edge strands that normally protect β-sandwich proteins from self-association. The results demonstrate that aggregation pathways can involve self-assembly of highly native-like folding intermediates, and have implications for the prevention of this, and other, amyloid disorders.

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Figure 1: Kinetic elucidation of the folding mechanism of wild-type β2m and P32G.
Figure 2: Amyloid-fibril formation from wild-type β2m and P32G at pH 7.0 and 37 °C.
Figure 3: Identification of conformational-exchange processes in P32G.
Figure 4: Analysis of 1H-15N HSQC spectra of wild-type β2m and P32G at pH 7.0, 37 °C.
Figure 5: Schematic free-energy landscape of wild-type β2m.

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Acknowledgements

We thank A.P. Kalverda, S.L. Myers, S. Jones and I.J. Morten for their help and support throughout this work, M.B. Pepys, G.A. Tennent, R. Wetzel and J.D. Fryer for kindly providing materials used and C.M. Dobson, F. Chiti and members of S.E.R.'s and S.W.H.'s group for helpful discussions. T.R.J. was supported by the Wellcome Trust, M.J.P. is a Biotechnology and Biological Sciences Research Council David Phillips Fellow and University of Leeds Research Fellow and S.E.R. is a Biotechnology and Biological Sciences Research Council Professorial Fellow.

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Authors and Affiliations

  1. Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK

    Thomas R Jahn, Martin J Parker, Steve W Homans & Sheena E Radford

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  1. Thomas R Jahn
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Correspondence to Sheena E Radford.

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Supplementary information

Supplementary Fig. 1

Double-jump refolding experiment for wild-type β2m. (PDF 133 kb)

Supplementary Fig. 2

Double-jump unfolding kinetics for wild-type β2m. (PDF 333 kb)

Supplementary Fig. 3

Global analysis of the folding and unfolding kinetics of wild-type β2m. (PDF 183 kb)

Supplementary Fig. 4

Global analysis of the folding and unfolding kinetics of P32G β2m. (PDF 345 kb)

Supplementary Table 1

Parameters for wild-type β2m folding kinetics (PDF 65 kb)

Supplementary Table 2

Parameters for P32G β2m folding kinetics (PDF 58 kb)

Supplementary Methods

Methods used for double-jump experiments and kinetic modeling (PDF 97 kb)

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Jahn, T., Parker, M., Homans, S. et al. Amyloid formation under physiological conditions proceeds via a native-like folding intermediate. Nat Struct Mol Biol 13, 195–201 (2006). https://doi.org/10.1038/nsmb1058

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