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Issue 45, 2019
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Tracking amyloid oligomerization with monomer resolution using a 13-amino acid peptide with a backbone-fixed spin label

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Abstract

Amyloid oligomers are suspected as toxic agents in neurodegenerative disease, and are transient and often heterogeneous, making them difficult to detect. Here we show an approach to track the development of amyloid oligomers in situ by room temperature, continuous wave (cw) 9 and 95 GHz EPR. Three amyloid peptides with the 2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid (TOAC) spin label were synthesized by solid phase peptide synthesis: T0EZ (TKVKVLGDVIEVGG) with TOAC (T) at the N-terminus, T5EZ with TOAC in the middle (KVKVTGDVIEVG) and T12EZ with TOAC at the C-terminus (KVKVLGDVIEVTG). These sequences are derived from the K11V (KVKVLGDVIEV) amyloid peptide, which self-aggregates to oligomers with a β-sheet configuration (A. Laganowsky, et al., Science, 2012, 335, 1228–1231). To monitor oligomerization, the rotational correlation time (τr) is measured by cw-EPR. For the backbone-fixed TOAC label that is devoid of local mobility τr should reflect the rotation and thereby the size of the peptide, resp. oligomer. For T5EZ a good match between the measured τr and the size of the peptide is obtained, showing the validity of the approach. One of the three peptides (T0EZ) aggregates (circular dichroism), whereas the other two do not. Since also the respective MTSL (S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate) labelled peptides fail to aggregate, molecular crowding due to the label, rather than the helix-inducing properties of TOAC, seems to be responsible. Following in situ oligomer formation of T0EZ by the change in rotational correlation time, two oligomers are observed, a 5–6 mer and a 15–18 mer. The EPR approach, particularly 95 GHz EPR, enables following oligomerization of one monomer at a time, suggesting that the cw-EPR approach presented is a novel tool to follow amyloid oligomerization with high resolution.

Graphical abstract: Tracking amyloid oligomerization with monomer resolution using a 13-amino acid peptide with a backbone-fixed spin label

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Publication details

The article was received on 01 Dec 2018, accepted on 24 Sep 2019 and first published on 07 Nov 2019


Article type: Paper
DOI: 10.1039/C9CP01060B
Phys. Chem. Chem. Phys., 2019,21, 25187-25195
  • Open access: Creative Commons BY-NC license
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    Tracking amyloid oligomerization with monomer resolution using a 13-amino acid peptide with a backbone-fixed spin label

    E. Zurlo, I. Gorroño Bikandi, N. J. Meeuwenoord, D. V. Filippov and M. Huber, Phys. Chem. Chem. Phys., 2019, 21, 25187
    DOI: 10.1039/C9CP01060B

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