Structural motif of polyglutamine amyloid fibrils discerned with mixed-isotope infrared spectroscopy - PubMed (original) (raw)

Structural motif of polyglutamine amyloid fibrils discerned with mixed-isotope infrared spectroscopy

Lauren E Buchanan et al. Proc Natl Acad Sci U S A. 2014.

Abstract

Polyglutamine (polyQ) sequences are found in a variety of proteins, and mutational expansion of the polyQ tract is associated with many neurodegenerative diseases. We study the amyloid fibril structure and aggregation kinetics of K2Q24K2W, a model polyQ sequence. Two structures have been proposed for amyloid fibrils formed by polyQ peptides. By forming fibrils composed of both (12)C and (13)C monomers, made possible by protein expression in Escherichia coli, we can restrict vibrational delocalization to measure 2D IR spectra of individual monomers within the fibrils. The spectra are consistent with a β-turn structure in which each monomer forms an antiparallel hairpin and donates two strands to a single β-sheet. Calculated spectra from atomistic molecular-dynamics simulations of the two proposed structures confirm the assignment. No spectroscopically distinct intermediates are observed in rapid-scan 2D IR kinetics measurements, suggesting that aggregation is highly cooperative. Although 2D IR spectroscopy has advantages over linear techniques, the isotope-mixing strategy will also be useful with standard Fourier transform IR spectroscopy.

Keywords: Huntington disease; antiparallel β-sheets; isotope dilution; two-dimensional infrared spectroscopy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Visualizations of fibril models used in MD simulations. Schematic of monomer conformation (Top), one-half of the fibril unit cell (Middle), and fibril cross-section (Bottom) for the (A) β-arc and (B) β-turn fibril models.

Fig. 2.

Fig. 2.

Transmission electron microscopy and experimental 2D IR spectra of Q24 fibrils. Electron micrographs of (A) unlabeled and (B) 13C-labeled Q24 fibrils. In each image, the white scale bar represents 200 nm. Two-dimensional IR spectra and diagonal intensity slices of (C) unlabeled and (D) 13C-labeled Q24 fibrils. (E) Polarization difference spectrum of 13C-labeled Q24 fibrils. Diagonal peaks are labeled BB for backbone modes and M for mixed backbone–side-chain modes. Symmetry labels are added for backbone modes in the difference spectrum. Cross-peaks between diagonal modes are highlighted with boxes. Frequencies are summarized in

Table S1

.

Fig. 3.

Fig. 3.

Simulated 2D IR spectra of Q24 fibril models. Simulated 2D IR spectra and diagonal intensity slices for the (A) β-arc and (B) β-turn models of unlabeled Q24 fibrils. Diagonal peaks are labeled BB for backbone modes, M for mixed backbone–side-chain modes, and TT for modes from the disordered turns and termini. Cross-peaks between the BB and M modes are highlighted with boxes. Frequencies are summarized in

Table S1

.

Fig. 4.

Fig. 4.

Experimental and simulated 2D IR spectra of isotope diluted fibrils. (A) Experimental 2D IR spectrum and diagonal intensity slice of 10% 13C-labeled Q24 fibrils. Simulated 2D IR spectra and diagonal intensity slices for (B) β-arc and (C) β-turn models of ∼8% labeled Q24 fibrils. Diagonal peaks are labeled with BB for backbone modes, M for mixed backbone–side-chain modes, and TT for disordered modes from the turns and termini. Peaks arising from 13C-labeled peptides are highlighted with boxes. Peak frequencies are summarized in

Table S2

.

Fig. 5.

Fig. 5.

Kinetics of Q24 aggregation. Difference slices obtained from 2D IR spectra of 13C-Q24. The initial slice (red) was subtracted from subsequent slices to highlight changes.

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