Calcium improves the overall positive charge of ClyF with minor effects on its secondary structure. (a) Dose-dependent enhancement of calcium on the bactericidal activity of ClyF against S. aureus N315. (b) Surface contact potential analysis of ClyF by PyMOL. The calcium-binding motif is marked with dashed oval. Negatively charged regions are labelled in red, while positively charge regions are in blue. (c) Concentrations of calcium bound to ClyF under different conditions. ClyF was mixed with 0, 0.1 and 1 mM CaCl2 for 1 h at room temperature and dialysed against 20 mM Tris. Concentrations of calcium in resulting samples were determined by colorimetric assay. (d–f) Effects of calcium, magnesium and zinc on the non-specific binding of ClyF to nucleic acids. Purified pET28a(+) plasmid was mixed with 0, 0.1, 1 and 5 mM CaCl2 (d), ZnCl2 (e) and MgCl2 (f) in the presence or absence of 2.5 µM ClyF for 1 h at room temperature, and then underwent electrophoresis on 1% agarose gel. The first lane on the left is the standard DNA ladder. The retention band and size of pET28a(+) are marked with triangles. (g–i) Circular dichroism spectra of ClyF under different concentrations of CaCl2, ZnCl2 and MgCl2. Spectra of ClyF in PBS (pH 7.4) containing 0.1, 1, 5 and 10 mM CaCl2 (g), ZnCl2 (h) and MgCl2 (i) were obtained by scanning with a circular dichroism spectrometer from 190 to 260 nm at room temperature and data from 200 to 260 nm are shown. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.
