Zinc Binding Assay for FrmR Variants

Experiments were carried out as described previously (50 (link)). FrmR, FrmR^E64H, FrmR^C35A, or FrmR^H60L was incubated (120 min) with an excess of ZnCl₂ in 100 mm NaCl, 400 mm KCl, 10 mm HEPES, pH 7.0, and an aliquot (0.5 ml) was resolved by size exclusion chromatography (PD10 Sephadex G25, GE Healthcare) in the same buffer conditions. Fractions were analyzed for zinc by inductively coupled plasma MS and for protein by a Bradford assay as described (50 (link)). The control experiments with FrmR and FrmR^E64H (Fig. 4E) are new unpublished data sets and are presented here to demonstrate reproducibility and for comparative purposes.