Probing metal ion substrate-binding to the E. coli ZitB exporter in native membranes by solid state NMR

Figures & data

Table I.  Primer sequences used in the construction of expression plasmids.

Name	Sequence
ZitB-1-F	5′ GCTGCCCGGGCTGCTGCTAACATCACCATCACCACC 3′
ZitB-1-R	5′ GGTGGTGATGGTGATGTTAGCAGCAGCCCGGGCAGC 3′
ZitB-F	5′ CACCGAATTCTGCGCACTCACACTCAC 3′
ZitB-R	5′ AAGCCTGCAGTTAATGGTGATGATGTGAATGG 3′
C230S-F	5′ AAGCGTCGTATGTCCCGGGAAATCCCGGAAGTCCGCA 3′
C230S-R	5′ TGCGGACTTCCGGGATTTCCCGGGACATACGACGCTT 3′
C294S & C299S-F	5′ CAACCTTCTCATGGGCCAGACTCGCACCTGAATGAAG 3′
C294S & C299S-R	5′ ATTCAGGTGCGAGTCTGGCCCATGAGAAGGTTGAT 3′

Figure 1.  Expression levels and tag-based detection of over-expressed proteins in membrane samples. SDS-PAGE fractionation of membrane samples with detection of protein by Coomassie blue staining (CB); biarsenical detection of fluorescence due to the Lumio™ tag (Lumio); India™ probe detection of His₈ tag (His-tag). In each case the arrowhead indicates the protein of interest. (a) ZitB(TEV-Lumio™-His₈) with detection of the His₈ tag; (b) ZitB(TEV-Lumio™) with detection of the Lumio tag; (c) Cysteineless ZitB carrying the mutations C230S, C294S, C299S and lacking the Lumio™-tag shown by Coomassie staining of protein in IPTG induced (I) compared with an uninduced (U) control; (d) GltP(TEV-Lumio™-His₈) with detection of the His-tag. In all cases substantial bands are visible in the Coomassie blue stained gel indicating good over-expression of protein.

Figure 2.  ¹¹³Cd CP-MAS analysis of membranes from E. coli expressing the zinc transporter ZitB(TEV-Lumio™-His₈). (a) ¹¹³Cd CP-MAS spectrum upon incubation with 5 mM [¹¹³Cd]CdCl₂ and (b) following preincubation with 50 mM ZnCl₂. Spectra were recorded at 66.4 MHz for ¹¹³Cd using a MAS frequency 2.4 kHz, CP contact time 7 msec and acquisition delay 1 sec over 28 000 scans at a temperature of 4°C.

Figure 3.  ¹¹³Cd CP-MAS analysis of E. coli membranes. ¹¹³Cd CP-MAS spectra were obtained by incubation of 5 mM [¹¹³Cd]CdCl₂ with E. coli membranes containing ZitB(TEV-Lumio™) before (a) and after (b) incubation with 50 mM ZnCl₂ or containing cysteineless ZitB (no tag) before (c) and after (d) incubation with 50 mM ZnCl₂ or containing (e) GltP(His₈). NMR conditions as in Figure 2.

Figure 4.  Transport of metal ions by reconstituted ZitB. Changes in Fluozin-1 fluorescence on metal ion addition (arrow) to proteoliposomes compared with control liposomes. ΔF/ΔFmax is relative fluorescence change normalized to maximum fluorescence on proteoliposome dissolution. (a) Zn^2 + transport into proteoliposomes at 1:325 (w/w) and 1:81 (w/w) ratio of ZitB to lipid. For (b) and (c) a 1:81 ratio was used. (b) Ni^2 + and Cd^2 + transport into proteoliposomes (c) competition assay showing that Cu^2 + ions inhibit the transport of Cd^2 + ions.