Fig. 1: Relative metal preferences of MncA folded in competing buffered metals.
a The Irving-Williams order of formation of complexes with metals, as the ionic states of exchangeable metals in the cytosol. From weak (left) to tight, noting reversal of arrow after copper. b Protocol for recovery of unfolded MncA from inclusion bodies after high-level expression in E. coli BL21(DE3) pLysS for 3 h using pET29aMncA at 37 °C, followed by folding denatured MncA via dropwise dilution into a large volume of urea-free buffer containing pairs of buffered and competing metal ions. Folded MncA was concentrated by anion exchange and eluted with single-step high-salt buffer. Metal-bound MncA was separated from unbound metal by SEC (PD-10 column), and metal analysed by ICP-MS. Gel inset shows, from left, size markers (from top, 100, 70, 55, 35, 25, 15 kDa), overloaded urea-solubilised MncA and MncA eluted from Q-Sepharose (full gel Supplementary Fig. 2a). c Metal contents of MncA-containing fractions after folding in excesses of competing metals buffered to different ratios of availabilities (as in panel b, buffers in Supplementary Table 1 prepared with assistance from Supplementary Data 1). Ratios of competing metals above each panel. The concentration of MncA approximated from A280nm (ɛ = 120,000 M−1). Stoichiometries approximate 2:1 but proportional metalation was calculated from total metal as in Table 1. Experiments with CuI and FeII performed in an anaerobic chamber, metal stocks prepared freshly, hydroxylamine (1 mM) used to sustain reduced copper. FeII and CuI confirmed to be > 95% reduced via reaction with excess ferrozine or BCA respectively. n = 3 independent experimental replicates shown. d Quantified preferences of MncA for different divalent metals, plus monovalent copper, relative to MnII (n = 3 independent experiments, triangles, squares, and circles depict replicates in (c), (I) and (II) denote forms of copper), determined from the elution profiles in (c). The order of preferences follows the Irving-Williams series as in (a). MncA prefers all non-cognate metals over MnII. Source data are provided as a Source Data file.
