Amyloid Assembly Endows Gad m 1 with Biomineralization Properties

Abstract

Acid proteins capable of nucleating Ca²⁺ and displaying aggregation capacity play key roles in the formation of calcium carbonate biominerals. The helix-loop helix EF-hands are the most common Ca²⁺-binding motifs in proteins. Calcium is bound by the loop region. These motifs are found in many proteins that are regulated by calcium. Gad m 1, an Atlantic cod β-parvalbumin isoform, is a monomeric EF-hand protein that acts as a Ca²⁺ buffer in fish muscle; the neutral and acid apo-forms of this protein can form amyloids. Since Ca²⁺-nucleating proteins have a propensity to form extended β-strand structures, we wondered whether amyloid assemblies of an EF-hand protein were able to influence calcium carbonate crystallization in vitro. Here, we used the Gad m 1 chain as a model to generate monomeric and amyloid assemblies and to analyze their effect on calcite formation in vitro. We found that only amyloid assemblies alter calcite morphology.

Keywords: EF-hand motif; Gad m 1; amyloids; calcite; calcium carbonate precipitation.

Figure 1

Structural features of the Gad m 1 chain. (A) Gad m 1 sequence and its dual folding signatures. Helical segments A–F forming the helix-loop-helix (AB, CD and EF) of EF-hands are depicted by colored cylinders. Acid loops binding Ca²⁺ and joining C and D and E and F helices are underlined by a thick blue line. Regions forming the aggregated core of the amyloid fold are underlined by a thick red line. Residues used for mutant generation (I12C and C19S) are outlined in cyan. (B) Three-dimensional structure of Ca²⁺-bound Gad m 1 (PDB 2MXY). Helical segments are depicted using the color code used in panel A, and cations are depicted as green spheres. (C) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel of the recombinant Gad m 1 wild type (wt), I12C and C19S chains obtained after cleavage of the His-tag.

Figure 2

Conformational features of the Ca²⁺-bound Gad m 1 wt and mutant chains. (A) Far-ultraviolet (UV) CD spectra of Ca²⁺-bound Gad m 1 chains depicting their helical fold. (B) Thermal denaturation of Ca²⁺-bound Gad m 1 chains displaying the cooperativity and stability of their folds. Denaturation curves were obtained from the changes in molar ellipticity at 222 nm that occurred upon heating. (C) Hydrodynamic radius of Gad m 1 chains derived from DLS measurements with an average polydispersity of 5–10%. The label wt ethylenediaminetetraacetic acid (EDTA) corresponds to Gad m 1 wt treated with 5 mM EDTA, which was included as a folding control. R_H: hydrodynamic radii, θmrw: mean residue weight ellipticity.

Figure 3

Amyloid aggregation of Gad m 1 chains. (A) Kinetics of amyloid aggregation monitored by thioflavin T (ThT) fluorescence. (B) CD spectra of Gad m 1 aggregates isolated by ultracentrifugation. (C) Dot-blot analysis of the recognition of the aggregation reaction products by anti-amyloid fibril (OC) and anti-amyloid oligomer (A11) antibodies. The label B corresponds to the Ca²⁺-bound Gad m 1 wt monomer.

Figure 4

Atomic force micrographs of Gad m 1 amyloid assemblies. Images of the aggregation products of Gad m 1 (A) wt, (B) I12C, (C) C19S and (D) wt in presence of tris(2-carboxyethyl)phosphine hydrochloride (TCEP). Height histograms correspond to the full images.

Figure 5

Stability of amyloid aggregates under calcium carbonate crystallization conditions. Amyloid dissociation was determined as the percentage of thioflavin T (ThT) fluorescence remaining after 20 h incubation in 0.1 M CaCl₂.

Figure 6

Scanning electron microscopy (SEM) images of the calcium carbonate precipitates obtained in vitro. Precipitates were obtained in the (A) absence and presence of Gad m 1 (B) wt monomer, (C) wt amyloids, (D) I12C amyloids and (E) C19S amyloids. The protein concentration was 0.2 mg/mL.

Figure 7

Representative (A) Transmission electron microscopy (TEM) image and the corresponding (B) Selected area electron diffraction (SAED) pattern from the marked area of the crystal along the [010] projection of the powder calcium carbonate precipitates obtained in the presence of Gad m 1 wt and mutant amyloids.