AFM images of monomeric filaments, oligomers, and protofibrils. Superposition of AFM phase and height images of various intermediates and late stage aggregates of lysozyme amyloid growth. (a) Mixture of monomeric filaments (orange) and taller, wider mature fibrils (green) grown in 50 mM NaCl at pH = 2 and T = 50 °C. Both fibril populations are very straight and mechanically stiff. (b) Early-stage oligomeric intermediates (orange/green) and (c) late stage protofibrils (green) obtained during incubation at 175 mM NaCl. Protofibrils are much more curvilinear despite being noticeably thicker than the stiff monomeric filaments shown in (a). A detailed analysis of all intermediates and their morphological features were presented in Ref. 15 . Scale bars represent 1 μm in (a) and (c) and 250 nm in (b). False color height scales are identical in all images.
Infrared spectra of late stage fibrils in either pathway show characteristic β-sheet peaks. Amide I peak for (a) native lysozyme in buffer at pH 2.0, and for mature fibrils analyzed after ultracentrifugation and incubation under (b) oligomer-free or (c) oligomeric growth conditions. Black lines represent the raw spectra while gray lines indicate the gaussian decomposition of the spectra into α-helix, β-turn, and β-sheet peaks as well as the resulting overall fit to the raw spectra. The bar graphs to the right of each spectrum represent the relative amplitudes of the α-helix and β-sheet peaks derived from spectral decomposition.
Difference spectra for lysozyme solution undergoing oligomeric fibril growth. (a) Renormalized amide I peak for monomeric lysozyme at pH 2 (solid black curve) and for late-stage fibrils grown along either the oligomer-free (solid red curve) or oligomeric pathway (solid blue curve). (b) Corresponding FTIR difference spectra of the Amide I region for oligomer-free fibrils (red curves) and for oligomeric protofibrils (blue curves) at pH = 2.0. The two difference spectra for oligomer-free fibril growth are for samples either incubated for multiple days (solid red curve) or derived from rapid growth (<12 h) via seeding with pre-formed fibrils (short-dashed red curve). Similarly, oligomeric difference spectra are shown for growth with 175 mM NaCl (solid blue curve), during rapid growth (<3 h) in 325 mM NaCl (short-dashed blue curve), or for growth at pH 7/ 65 °C (blue dotted curve) (c) Temporal evolution of the Amide I difference spectrum for lysozyme undergoing oligomeric fibril growth.
Rate of lysozyme hydrolysis during oligomer-free vs. oligomeric fibril growth. Lysozyme monomers (1 mg/ml) were incubated for a total of 120 h under either oligomer-free (50 mM NaCl) or oligomeric (175 mM NaCl) fibril growth conditions (pH 2, 507 °C). Aliquots were removed at various time points and analyzed with SDS PAGE electrophoresis under either (top) non-reducing or (bottom) reducing conditions (1 μl β-mercaptoethanol).
ThT fluorescence, light scattering intensity, and particle size distributions during oligomer-free fibril growth. (a) Fractional changes in ThT fluorescence intensity (■) and light scattering intensity (□) measured for lysozyme undergoing oligomer-free filament growth (50 mM NaCl, pH 2, 50 °C). The sudden increase in both signals coincides with the nucleation of monomeric filaments shown below. (b) Temporal evolution of the particle size distribution (PSD) for the same sample. Each point represents the dominant hydrodynamic radius for separable peaks in the PSD obtained from DLS measurements. During an extended lag period a single peak near the monomeric radius of lysozyme (Rh = 1.9 nm) persists until two additional peaks emerge with maximal radii near 400 nm and 40 nm, respectively. This nucleation event coincides with the appearance of monomeric filament populations in AFM images with lengths distributions consistent with the two DLS peaks.
ThT fluorescence, light scattering intensity, and particle size distributions during oligomeric fibril growth. (a) Fractional changes in ThT fluorescence intensity (■) and light scattering intensity (□) measured from lysozyme undergoing oligomeric fibril growth (175 mM NaCl, pH 2, 50 °C). Both ThT fluorescence and light scattering intensity show a continuous, accelerating increase with no discernible signature of the prominent nucleation event detected by DLS. (b) Dominant hydrodynamic radii for separable peaks in particle size distributions obtained during oligomeric fibril assembly (175 mM NaCl). The subtle upward drift in the monomer peak results from the formation of small populations of oligomers readily seen in AFM. The new peak emerging around 15 h indicates the nucleation of protofibrils formed from oligomers.
Evolution of aggregate peaks during oligomer-free vs. oligomeric fibril growth. Particle size distributions (PSDs) with DLS for either (a) oligomer-free filamentous growth at 50mM NaCl or (b) oligomeric fibril assembly at 175 mM NaCl. The gray bars represent the PSD obtained shortly after (a) nucleation of the two populations of rigid filaments (short and long filaments) or (b) nucleation of protofibrils. The empty bars, in turn, represent the corresponding PSDs near the end of the measurement period for either pathway.
Correlation of ThT fluorescence and light scattering in different assembly pathways. (a) Ratio of fractional ThT over fractional light scattering increments for late-stage fibrils vs. the NaCl concentrations used during their growth. ThT/light scattering ratios for fibrils after oligomer-free (<150 mM NaCl) vs. those after oligomeric fibril growth (>150 mM NaCl) differ by approximately two orders of magnitude (note logarithmic scale). The onset of amorphous precipitation near 400 mM NaCl causes another sudden drop in this ratio by a factor of ten. (Inset) Salt-dependent changes in ThT fluorescence emission (■) in presence of lysozyme, (◯) for ThT without lysozyme and (□) for lysozyme auto-fluorescence, all measured at 485 nm. (b) Log-log plot of the fractional changes in ThT fluorescence vs. their corresponding fractional light scattering intensity as they evolve during the entire aggregation process. Results are shown for multiple lysozyme samples incubated under conditions of either monomeric filament growth (solid symbols) or oligomeric fibril growth (open symbols). The dashed lines represent linear fits through data at a fixed salt concentration. ThT ratios display strong and persistent linear correlations to their corresponding light scattering ratios throughout the entire growth process. (c) ThT and light scattering ratios measured for fibril growth of lysozyme at physiological pH (pH 7, 20 mM NaCl, T = 65 °C).
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