In vitro crystallization experiments in the presence of ubiquitylated proteins.

The effect of ubiquitylated proteins on the growth of calcium carbonate crystals was tested in two crystallization systems, i.e., calcite growth in the left column and aragonite growth in the middle column. (B) 30 µg/ml BSA was added as the negative control. (F) 30 µg/ml EDTA-soluble matrix of the prismatic layer was added as the positive control. The crystals were etched. (I) 30 µg/ml ubiquitylated proteins were added to the system to induce the formation of vaterite. (N) 30 µg/ml IAC unbound fraction was added. (D), (H), (L), and (P) are the same as those shown in the left column, except that 50 mM Mg²⁺ was added to these systems. (H) No crystals were formed in the aragonite growth systems, when EDTA-soluble matrix of the prismatic layer was added. (L) The ubiquitylated proteins induced calcite formation at a 30 µg/ml concentration. (P) The IAC unbound fraction could induce the formation of round crystals. Right column, Raman spectra of the crystals formed in the left and middle columns. (A), (E), (I), (M), (C), (G), (K), and (O) show enlarged images of the boxed regions in (B), (F), (J), (N), (D), (H), (L), and (P) respectively. Scale bars: 100 µm in (F), (J), (L), (N) and (P); 50 µm in (D) and (H); 25 µm in (B); 10 µm in (I) and (K); 5 µm in (A), (C), (E), (G), (M) and (N).