Figure 3
Bioinformatics and biochemical characterisation of ZnuA.
(a) Absorbance (280 nm) trace of ZnuA analysed by gel permeation chromatography. Inset represents linear regression of soluble molecular mass standards used to determine the apparent molecular mass of ZnuA. ZnuA was determined to be monomeric with a molecular mass of 34.5 kDa. (b) Coomassie-stained SDS-PAGE analysis of monomeric ZnuA (indicated by arrow) by comparison with soluble molecular mass standards. Fractions analysed were 0.5 mL, with elution volume at start of each fraction indicated above the lane. (c) Cartoon representation of the homology-based model of P. aeruginosa ZnuA showing a typical cluster A-I fold and the predicted Zn2+ binding residues. (d) Primary sequence alignment of P. aeruginosa ZnuA and ZnuA orthologs from other bacterial species with the His-rich region indicated in blue. ZnuA proteins from: Pae, P. aeruginosa (GI:15600691); Hin, H. influenza (GI:491963406); Yru, Y. ruckeri (GI:490857750); Sen, S. enterica (GI:541470409); Eco, E. coli (GI:635897169); Tpa, T. palladium (GI:504108253); Cje, C. jejuni (GI:504330062); Syn, Synechocystis 6803 (GI:499174152). (e) Competitive binding experiment with apo-ZnuA using Mag-fura-2-Zn2+. The normalised fluorescence emission (520 nm) of Mag-fura-2 was monitored in response to the addition of increasing concentrations of apo-ZnuA. Data are the mean (±s.e.m.) for three independent experiments.
