Supplementary Figure 4: Functional analysis of mutant LpCopA forms.

(a), Purity of the assayed constructs used for the in vitro assay. SDS-PAGE gels of the purified LpCopA constructs following a first round of scaling using ImageJ. Identical relative amounts of protein were used for the Baginski assay²⁵ for generating the raw data shown in Supplementary Table 1. (b-c), The in vivo copper susceptibility assay. E. coli growth complementation curves from three independent experiments for wild-type LpCopA (WT, red), the inactive Asp426Asn mutant (gray), the high-affinity coordinator mutant Met717Val (yellow) as well as various release pathway mutants are displayed. Experiments 1 and 3 used three replicates each, experiment 2 used 15 replicates. 0 and 3 mM copper ((b) and (c), respectively) were used in the growth medium. (d), Assessment of the reproducibility of the in vitro assay. The in vitro data shown in Fig. 4c are based on a single experiment (experiment 1, with nine replicates), so wild type, Asp426Asn, Ala714Thr and Met100Glu LpCopA were reproduced in additional experiments (experiment 2, with six replicates). The two independent sets of data are highly consistent (merged data).