Pressure-dissociable reversible assembly of intrinsically denatured lysozyme is a precursor for amyloid fibrils

Abstract

Although a diversity of proteins is known to form amyloid fibers, their common mechanisms are not clear. Here, we show that an intrinsically unfolded protein (U), represented by a disulfide-deficient variant of hen lysozyme with no tertiary structure, forms an amyloid-like fibril after prolonged incubation. Using variable pressure NMR along with sedimentation velocity, circular dichroism, and fluorescence measurements, we show that, before the fibril formation, the protein forms a pressure-dissociable, soluble assemblage (U'(n)) with a sedimentation coefficient of 17 S and a rich intermolecular beta-sheet structure. The reversible assemblage is characterized with a Gibbs energy for association of -23.3 +/- 0.8 kJ.mol(-1) and a volume increase of 52.7 +/- 11.3 ml.mol(-1) per monomer unit, and involves preferential interaction of hydrophobic residues in the initial association step. These results indicate that amyloid fibril formation can proceed from an intrinsically denatured protein and suggest a scheme N <==>U <==>U'(n)-->fibril as a common mechanism of fibril formation in amyloidogenic proteins, where two-way arrows represent reversible processes, one-way arrow represents an irreversible process, and N, U, and U'(n)represent, respectively, the native conformer, the unfolded monomeric conformer, and the soluble assemblage of unfolded conformers.

Fig. 1.

Association of the 0SS variant. (A) A Schlieren pattern, taken 42 min after the start of sedimentation. Protein concentration was 7.6 mg·ml^–1 in 50 mM sodium maleate (pH 2.0). (B) Far-UV CD spectra of the 0SS solution incubated for 1 day at 25°C in 5 mM sodium maleate (pH 2.7)/45 mM sodium chloride at protein concentrations of 1, 2, 3, and 4 mg·ml^–1 (labeled on each spectrum).

Fig. 2.

(A) Thioflavine T fluorescence spectra of the 0SS solution incubated for (from bottom to top) 0, 1, 2, and 9 days, and 9 months at 25°C, in 50 mM sodium maleate (pH 2.0) at 8.0 mg·ml^–1 protein concentration. (B) Electron microgram of the 0SS fibrous aggregate formed after 8 months of incubation in 20 mM sodium acetate (pH 4.0) at a protein concentration of 2.5 mg·ml^–1. (Scale bar, 100 nm.) The fraction of thin and curved fibrils was higher in sodium maleate buffer than in sodium acetate buffer.

Fig. 3.

¹⁵N/¹H-HSQC spectra of uniformly ¹⁵N-labeled 0SS variant at various pressures indicated. Spectra at 30, 400, (800, 1,200, and 1,600 not shown), and 2,000 bars were taken at an increasing pressure cycle, and those at 600, 300, and 200 bars were taken at a decreasing pressure cycle. A total of 55 cross peaks were assigned. The cross peaks labeled in the spectrum at 600 or 400 bars diminish conspicuously with decreasing pressure as detailed in Fig. 5.

Fig. 4.

Variation in NMR peak volumes with pressure and thermodynamic analysis of the dissociation reaction. (A) Relative peak volumes of 47 individual residues as a function of pressure, calculated from ¹⁵N/¹H-HSQC spectra. Open circles represent mean values at each pressure. (B) Free-energy change for dissociation versus pressure, calculated for the association reaction of the order n = 33. The line was obtained by regression of the data within 10–90% of the transition width.

Fig. 5.

Relative HSQC peak volumes of 47 residues at 300 bars plotted against residue positions. The dotted horizontal line indicates the averaged value (0.134) over all of the assigned peaks. For the 11 residues labeled, the relative volume is <0.014 (filled short bars). The horizontal bars at bottom labeled with roman numerals indicate the regions for the six hydrophobic clusters reported in figure 2B of ref. (each bar corresponds to the residue region spanned by the half-height-width of each Gaussian distribution modeled for R₂ relaxation rates).