Collapsed state of polyglutamic acid results in amyloid spherulite formation

Abstract

Self-assembly of proteins and peptides into amyloid fibrils involves multiple distinct intermediates and late-stage fibrillar polymorphs. Understanding the conditions and mechanisms that promote the formation of one type of intermediate and polymorph over the other represents a fundamental challenge. Answers to this question are also of immediate biomedical relevance since different amyloid aggregate species have been shown to have distinct pathogenic potencies. One amyloid polymorph that has received comparatively little attention are amyloid spherulites. Here we report that self-assembly of the intrinsically disordered polymer poly(L-glutamic) acid (PLE) can generate amyloid spherulites. We characterize spherulite growth kinetics, as well as the morphological, optical and tinctorial features of this amyloid polymorph previously unreported for PLE. We find that PLE spherulites share both tinctorial and structural characteristics with their amyloid fibril counterparts. Differences in PLE's molecular weight, polydispersity or chemistry could not explain the selective propensity toward either fibril or spherulite formation. Instead, we provide evidence that PLE polymers can exist in either a collapsed globule or an extended random coil conformation. The collapsed globule consistently produces spherulites while the extended coil assembles into disordered fibril bundles. This results suggests that these 2 PLE conformers directly affect the morphology of the resulting macroscopic amyloid assembly.

Keywords: amyloid; birefringence; polyglutamic acid; polymer conformation; polymorphism; spectroscopy.

Figure 1.

Morphology of Sigma vs. Alamanda PLE Aggregates. Light and transmission electron microscopy (TEM) images of aggregates grown from (A-C) Alamanda PLE vs. (D-F) Sigma PLE at 1 mg/mL, pH 3.6, 50 mM citrate buffer and 40 °C. Bright field image of (A) spherical Alamada PLE aggregates vs. (D) disorganized Sigma PLE aggregates. (B, E) Thioflavin T fluorescence images of same aggregates as in (A) and (D), respectively. (C, F) TEM images of Alamanda vs. Sigma PLE aggregates grown under the same conditions.

Figure 2.

Optical Microscopy and IR Spectroscopy of PLE Spherulites (A) Same spherulites as in (Fig. 1D, E) imaged between crossed polarizers which reveals the Maltese cross pattern characteristic of structured polymeric spherulites, (B) Unstained PLE spherulites display a very intense intrinsic fluorescence emission associated with amyloid formation (ex. 380 nm, em. 455 nm) (C) Amide I band of the FTIR spectra for Alamanda PLE spherulites (solid line), Sigma PLE bundled fibrils (dot-dash line) and Alamanda PLE monomers (dotted line). Peak amplitudes of spectra were normalized to same heights for comparison.

Figure 3.

Kinetics of PLE Spherulite Assembly: Light Scattering vs. ThT Fluorescence (A) Superposition of the fractional increases in light scattering intensity ΔI/I₀ vs. ThT fluorescence intensity ΔF/Fo during spherulite growth of PLE (30 kDa, 2 mg/mL) in 50 mM citrate at pH 4.1, 50 mM NaCI, incubated at T = 37 °C. The insert shows the same data on a semi-log plot which indicates the lack of a lag phase under these conditions (B) Plot of ΔI/I₀ from (A) against the corresponding number of peaks in the particle size distribution (PSD), as obtained from dynamic light scattering. The appearance of a prominent new aggregate peak around 6 hours coincides with the dramatic upswing in scattering intensity.

Figure 4.

Concentration-dependence of PLE Spherulite Assembly (A) Increase in ThT fluorescence emission at 485 nm (445 nm ex.) during spherulite assembly of Alamanda PLE (7.5 kDa) at the indicated PLE concentrations (40 °C, pH 3.6, 50 mM NaCI). Solid lines and t_lag/C parameters represent the results of fits through the data using the JMAK spherulite growth model (B) Kinetics traces similar to (A) for four identical samples of PLE at 0.5 mg/mL and 0.25 mg/mL, respectively, highlighting the high degree of reproducibility of spherulite kinetics (C) TEM image of nanoscale Alamanda PLE spherulites obtained at high driving force (PLE concentrations ≥ 2 mg/mL).

Figure 5.

pH Dependence of Spherulite Growth (A) Far UV circular dichroism (CD) spectra of Alamanda PLE (7.5 kDa, 1 mg/mL, 37 °C) as function of solution pH. Alamanda PLE undergoes the typical cooperative transition from an alpha-helix to a random-coil conformation between pH 4.5 and 5.5.^67,58 (B) Slow down in the kinetics of PLE spherulite growth as pH approaches the edge for the alpha-helix conformation (C, D) ThT fluorescence responses from 1 mg/mL solutions of 7.5 kDa PLE incubated at high salt concentrations either (C) in the random coil state (pH 6.0) or (D) near the edge of the alpha helical conformation (pH 4.5).

Figure 6.

Temperature Effects on Monomer Structure and Spherulite Growth (A) Self-assembly of PLE (0.5 mg/mL, pH 3.6, 50 mM NaCI) into spherulites monitored at three different temperatures using ThT (10 [μM) as indicator dye. (B) CD spectra of PLE (0.2 mg/mL, pH = 3.6) vs. temperature. CD spectra indicate a gradual transition from α-helix to random coil conformation as temperature increases.

Figure 7.

Globule vs. Random Coil Conformation of PLE underlying Transition in Growth Behavior (A) SDS-PAGE analysis of Alamanda vs Sigma PLE stocks of various average molecular weights. Overall polydispersity of Alamanda PLE, while reduced, was not significantly different from Sigma PLE. (B) FTIR spectra of Sigma vs. Alamanda PLE at pH 7 in deuterium oxide reveal no obvious chemical differences between either PLE stock. (C) In situ particle size distribution of Alamanda (60 kDa, solid line) vs. Sigma PLE (50–100 kDa, dotted line) dissolved at pH 7 and 400 mM NaCI. The hydrodynamic radius Rh of Alamanda PLE is more than a factor of two smaller than that of Sigma PLE. Extensive dialysis of Alamanda PLE nearly doubles its Rh (dashed-dotted line) (D) TEM images of Alamanda PLE aggregates obtained from dialyzed PLE monomers incubated at pH 3.6.