On the role of peptide hydrolysis for fibrillation kinetics and amyloid fibril morphology

Abstract

Self-assembly of proteins into amyloid-like nanofibrils is not only a key event in several diseases, but such fibrils are also associated with intriguing biological function and constitute promising components for new biobased materials. The bovine whey protein β-lactoglobulin has emerged as an important model protein for the development of such materials. We here report that peptide hydrolysis is the rate-determining step for fibrillation of β-lactoglobulin in whey protein isolate. We also explore the observation that β-lactoglobulin nanofibrils of distinct morphologies are obtained by simply changing the initial protein concentration. We find that the morphological switch is related to different nucleation mechanisms and that the two classes of nanofibrils are associated with variations of the peptide building blocks. Based on the results, we propose that the balance between protein concentration and the hydrolysis rate determines the structure of the formed nanofibrils.

Fig. 1. Fibrillation kinetics of WPI. (A) Time dependence of the ThT fluorescence at 485 nm for WPI solutions of 10–80 g l⁻¹ incubated at 90 °C. The circles show the measured data (average of three samples) and the lines represents the best fits to the Finke–Watzky model. Error bars are ±1 standard deviation. (B) Initial rates, taken as the slope of the linear fits (ESI Fig. S4†) as function of initial sample concentration (filled circles). The max rates derived from the Finke–Watzky fit are shown for comparison (open circles). The colors of the data points correspond to those in (A). The black line shows the linear fit of the initial rates (i.e. only the filled circles).

Fig. 2. Changes in pH during WPI fibrillation and pH-dependence of the fibrillation kinetics. (A) Changes in pH of 40 g l⁻¹ (blue) and 77 g l⁻¹ (red) WPI samples during fibrillation. (B) The data from panel (A) displayed as normalized (1 − [H⁺]) together with normalized ThT fluorescence intensity (485 nm). (C) The logarithms of the initial fibrillation rates (in h⁻¹) of 40 g l⁻¹ (blue) and 78 g l⁻¹ (red) WPI samples as function of pH. The 40 g l⁻¹ sample at pH 2.8 (open blue circle) was not included in the linear fit (see the ESI for details).

Fig. 3. Comparison of normalized ThT fluorescence intensity and the formation of peptides <10 kDa in samples of 40 and 70 g l⁻¹ initial WPI concentration, respectively.

Fig. 4. AFM images illustrating the morphologies of the fibrils formed at low and high initial WPI concentrations. (A) Straight and long fibrils formed in a 40 g l⁻¹ WPI sample. (B) Curved and short fibrils formed in a 80 g l⁻¹ WPI sample.

Fig. 5. Structural characteristics of straight (blue lines) and curved (red lines) fibrils compared to non-fibrillar WPI at pH 2 (green lines). The fibrils were purified by dialysis and all protein solutions were adjusted to similar protein concentrations based on absorbance at 280 nm and dry weight measurements. (A) FTIR spectra measured on dry protein films. Data are scaled to show similar peak absorbances in the amide I region. (B) Far-UV CD spectra of protein solutions. (C) Congo red absorbance spectra. The grey line is Congo red in solution without any protein. (D) Changes in Congo red absorbance spectra compared to the dye in absence of protein. (E) ThT fluorescence spectra with excitation wavelength = 440 nm. (F) Protein auto-fluorescence spectra with excitation wavelength = 375 nm. All solution measurements were performed at pH 2, except for the Congo red absorbance that was performed in PBS buffer, pH 7.4.

Fig. 6. Peptide building blocks of PNFs identified by MALDI-TOF. The top spectrum (red) is for curved fibrils formed at 80 g l⁻¹ and the bottom spectrum is for straight fibrils formed at 40 g l⁻¹. The fibrils of the samples were purified and dissolved in 8 M GuHCl before analysis. The numbers are the molecular weights (in Da) of the corresponding peptide fragments carrying a single charge.

Fig. 7. AFM images demonstrating the cross-seeding ability of the straight fibrils. (A) A sample with 77 g l⁻¹ WPI fibrillated for 76 h. (B) The same WPI solution as in (A) with 5% seeds of straight fibrils added before fibrillation.

Fig. 8. Schematic illustration of the proposed molecular mechanism for the morphology switch. The arrows indicate hydrolysis (1), nucleation (2) and fibril elongation (3). At low starting concentration, hydrolysis results in peptide fragments with a length distribution that, on average, is shorter than at high concentrations. This affects the structure of the fibril nuclei as longer peptide segments will be included and the C-terminal core segment will be incorporated to a higher degree at higher starting concentrations.