Figure 2.

Conformational change and self-assembly of BS in CaCl₂ solution. (A) The Ca²⁺ binding ability of BS demonstrated by the decreasing absorbance at 570 nm of the mixture (made of 1.5 mL of 100 mM NaHCO₃ (pH 8.7) and 300 μL of 20 mM Tris-HCl solution) in the presence of added BS with different concentration (0, 2.5, 5.0, 7.5, 10.0 mg/mL) after the addition of 1.5 mL of 100 mM CaCl₂ (pH 8.7). The Tris-HCl solution (with NaHCO₃ added but in the absence of BS) was used as a control. The stronger inhibition of precipitation of calcium carbonate, reflected by the reduced absorbance at 570 nm due to the inhibited calcium carbonate precipitation, indicated the higher Ca²⁺ binding activity. (B) CD spectra of aqueous BS solution before and after the addition of 5 mM CaCl₂, respectively. The positive absorbance at 195 nm and negative absorbance at 217 nm indicated the appearance of β-sheet structure. (C-D) AFM image of the protein-based structures formed in aqueous BS solution before (C) and (D) after pre-incubation with 10 mM CaCl₂ for 5 days.