Fourier transform infrared spectroscopy provides a fingerprint for the tetramer and for the aggregates of transthyretin

Abstract

Transthyretin (TTR) is an amyloidogenic protein whose aggregation is responsible for several familial amyloid diseases. Here, we use FTIR to describe the secondary structural changes that take place when wt TTR undergoes heat- or high-pressure-induced denaturation, as well as fibril formation. Upon thermal denaturation, TTR loses part of its intramolecular beta-sheet structure followed by an increase in nonnative, probably antiparallel beta-sheet contacts (bands at 1,616 and 1,686 cm(-1)) and in the light scattering, suggesting its aggregation. Pressure-induced denaturation studies show that even at very elevated pressures (12 kbar), TTR loses only part of its beta-sheet structure, suggesting that pressure leads to a partially unfolded species. On comparing the FTIR spectrum of the TTR amyloid fibril produced at atmospheric pressure upon acidification (pH 4.4) with the one presented by the native tetramer, we find that the content of beta-sheets does not change much upon fibrillization; however, the alignment of beta-sheets is altered, resulting in the formation of distinct beta-sheet contacts (band at 1,625 cm(-1)). The random-coil content also decreases in going from tetramers to fibrils. This means that, although part of the tertiary- and secondary-structure content of the TTR monomers has to be lost before fibril formation, as previously suggested, there must be a subsequent reorganization of part of the random-coil structure into a well-organized structure compatible with the amyloid fibril, as well as a readjustment of the alignment of the beta-sheets. Interestingly, the infrared spectrum of the protein recovered from a cycle of compression-decompression at pD 5, 37 degrees C, is quite similar to that of fibrils produced at atmospheric pressure (pH 4.4), which suggests that high hydrostatic pressure converts the tetramers of TTR into an amyloidogenic conformation.

FIGURE 1

(a) X-ray structure of wt TTR (PDBfile 1F41) obtained at 1.5-Å resolution, showing the four monomers. (b) Secondary-structure analysis of wt TTR by FTIR. Curve fitting of the deconvoluted amide I′ band of wt TTR (37°C, pD 7.5) with eight Gauss-Lorentz functions: 1and 8, nonnative, aggregated β-sheets; 2 and 3, turns; 4, α-helices; 5, random coil; and 6 and 7, β-sheets.

FIGURE 2

Temperature-induced unfolding of wt TTR. (a) FTIR spectra of 4% (w/w) wt TTR at pD 5.0 as a function of temperature increase (only selected temperatures are shown). (b) Relative intensities of wt TTR secondary-structure components as a function of increasing temperatures. The isolated symbols on the right correspond to the respective secondary-structure components after returning to 25°C.

FIGURE 3

High-pressure-induced unfolding of wt TTR at 37°C. (a) FTIR spectra of 4% (w/w) wt TTR at pD 7.5 as a function of increasing pressure at 37°C (only selected pressures are shown). Relative intensities of the secondary-structure components as a function of pressure at pD 7.5 (b) and at pD 5.0 (c). The isolated symbols on the right correspond to the respective secondary-structure components after returning to atmospheric pressure (turns are not shown).

FIGURE 4

High-pressure-induced unfolding of wt TTR at 5°C. (a) FTIR spectra of 4% (w/w) wt TTR at pD 7.5 as a function of increasing pressure at 5°C (only selected pressures are shown). (b) Relative intensities of the secondary-structure components as a function of pressure at pD 7.5 and at pD 5.0 (c). The isolated symbols on the right correspond to the respective secondary-structure component after returning to atmospheric pressure (turns are not shown).

FIGURE 5

Comparing the secondary-structure content of wt TTR after different treatments by FTIR and CD. (a) FTIR spectra of 4% w/w TTR in different oligomeric states: native state pD 5.0 (solid black line); after heat unfolding at pD 5.0 (dotted black line); after decompression at pD 5.0 (dashed line); after decompression at pD 7.5 (solid shaded line); fibrils of TTR grown at pH 4.4 (dotted shaded line). (Inset) Comparison between the spectra obtained immediately after decompression at pD 5.0 (solid line) and 18 h after returning to atmospheric pressure (dotted line). (b) CD spectra of 5 μM wt TTR at pH 7.5 (solid line), TTR fibrils grown at pH 4.4 (dotted line), and of 5 μM TTR incubated at 80°C (dashed line).