Protein staining

Fig. 7. Expression of N-MNKP-MBP in the presence of thioredoxin and in vivo loading with copper. E. coli cells transformed with N-MNKP-MBP and thioredoxin-containing plasmids were grown under standard conditions. Copper was added up to 500 ^M (+) to one of the cultures and the protein expression in control and copper-treated sample was induced with IPTG as described (48). Following affinity purification, the availability of Cys residues for labeling with fluorescent coumarin maleimide (CPM) and the amount of copper bound to the protein were determined. In this experiment, the (+) copper sample contained 5.19 mol Cu/mol protein. Similar results were obtained with N-WNDP-MBP fusion.

Fig. 8, or by atom absorption spectroscopy. The two procedures yield similar results, although care should be taken using the BCA assay, because the accuracy of this procedure may be affected by buffer composition, (e.g., by presence of imidazole (67; and our unpublished data).

Binding of copper to N-WNDP, N-MNKP, or to copper chaperones, such as HAH1, all of which contain the same CxxC motif for copper coordination, can be monitored by decrease of the Cys residues reactivity toward fluorescent reagents following copper binding to the protein, as shown in Fig. 7. Although this assay is indirect and has to be confirmed by the BCA-based analysis or by atom absorption spectroscopy, it could be very valuable when comparing multiple samples. The labeling assay is fairly independent on buffer composition and requires significantly less protein than the BCA-based procedure and atom absorption spectroscopy. We observed good agreement in copper stoicheometry values comparing the BCA procedure with the fluorescent probe-based assay, and we routinely used both protocols for the copper-binding measurements.

6.3. Choosing a Tag for Affinity Purification of the Copper-Binding Domain

For the characterization of the copper-binding domain of WNDP, we utilized the MBP fusion and a histidine tag. The advantage of the MBP fusion is that MBP does not bind copper and therefore does not appear to interfere with copper-binding properties of WNDP, in contrast to HisTag, which binds copper (Cu2+). The disadvantage of MBP fusion is its large size (42 kDa), which complicates structural analysis of the fusion protein. For the characterization of MerP, expressed as a MBP fusion, MBP was cleaved prior to spectroscopic analysis (47); however, trombin cleavage of N-WNDP-MBP, which is significantly larger than MerP-MBP, was fairly inefficient (our data). Therefore, for characterization of the N-WNDP secondary structure, we utilized a HisTagged version of this protein (Fig. 3).

Interestingly, N-WNDP-HT can be expressed in a soluble form in E. coli in the absence of thioredoxin, but it has to be reduced in vitro in order to get copper bound (Fig. 9).

Fig. 8. Measurements of copper-binding to N-WNDP (N-WND) and N-MNKP (N-MNK) using bicinchoninic acid. Proteins loaded with copper in vivo as in Fig. 7 were used to estimate the amount of bound copper.

Fig. 9. Comparative analysis of the in vitro copper-binding properties of N-WNDP-MBP and N-WNDP-HT. Left panel: N-WNDP-MBP was expressed in the presence of thioredoxin, purified using amylose resin, and loaded with copper in vitro in the presence of ascorbate as described in ref. 48. Copper-binding was monitored by labeling of Cys residues with fluorescent coumarin maleimide and confirmed with BCA assay. Right panel: N-WNDP-HT was expressed without thioredoxin, purified on NTA resin and was either eluted with Imidazole (first two lanes) or was incubated with or without copper in the presence of reducing reagent [tris-(2-carboxyethyl)phosphine hydrochloride, TCEP] while it is was bound to the resin. Note that without reduction, Cys residues are unavailable for labeling with the fluorescent probe. Following washes and elution with imida-zole, the amount of copper bound to N-WNDP-HT was determined as above.

Fig. 9. Comparative analysis of the in vitro copper-binding properties of N-WNDP-MBP and N-WNDP-HT. Left panel: N-WNDP-MBP was expressed in the presence of thioredoxin, purified using amylose resin, and loaded with copper in vitro in the presence of ascorbate as described in ref. 48. Copper-binding was monitored by labeling of Cys residues with fluorescent coumarin maleimide and confirmed with BCA assay. Right panel: N-WNDP-HT was expressed without thioredoxin, purified on NTA resin and was either eluted with Imidazole (first two lanes) or was incubated with or without copper in the presence of reducing reagent [tris-(2-carboxyethyl)phosphine hydrochloride, TCEP] while it is was bound to the resin. Note that without reduction, Cys residues are unavailable for labeling with the fluorescent probe. Following washes and elution with imida-zole, the amount of copper bound to N-WNDP-HT was determined as above.

Although the N-WNDP-HT is useful for analysis of copper-independent properties of this domain, such as overall folding, and structure, its usefulness for analysis of copper binding is somewhat ambiguous because of the ability of HisTag to bind copper. In fact, our initial attempts to load N-WNDP with copper either in vivo or in vitro led to protein precipitation. The precipitation problem can be avoided if copper is added to N-WNDP-HT during purification while protein is still bound to the NTA resin and the His tails are sequestered by interactions with Ni. This protocol allowed copper to bind to Cys residues, as shown in Fig. 9.

TNP-ATP, jiM

Fig. 10. The comparison of the nucleotide-binding properties for the ATP-binding domains of WNDP (filled circles) and Na+,K+-ATPase (the typical representative of the P2-type ATPases, empty circles). The identical amounts of purified nucleotide-binding domains were mixed with increasing concentrations of TNP-ATP and the nucleotide-binding was monitored by the increase in TNP-ATP fluorescence.

The column-based procedure yields a copper-bound N-WNDP with stoichiometry close to what was found for in vitro-loaded N-WNDP-MBP; however, there is a marked difference between N-WNDP-HT and N-WNDP-MBP in the stability of the copper-protein complex. Although copper is bound tightly to N-WNDP-MBP, such that copper-bound protein can be dialyzed or concentrated significantly without losing copper, the HisTag fusion of WNDP quickly loses its copper upon concentration and then begins to aggregate when protein concentration exceeds 1-2 mg/mL. We conclude that the MBP fusion expressed in the presence of thioredoxin and loaded with copper in vivo currently represents a much better system for characterization of N-WNDP and N-MNKP properties.

6.4. ATP-Binding Domain

The problems associated with the HisTag, which we discussed earlier, could be the result of the fact that both the HisTag and the N-terminal domain can bind copper, and the presence of two copper-binding motifs generates protein with completely new properties. Using the HisTag, however, works fairly well for expression and purification of the ATP-binding domain of WNDP (ATP-BD). Although solubility of this domain is rather limited, and is not improved by coexpression with thioredoxin, it is possible to obtain up to 500 ^g of purified protein from 2 L of cell culture following induction with 0.1 mM isopropyl-l-thio-^-D-galactopyranoside (IPTG) at room temperature (55).

The ability of the ATP-binding domain to bind the nucleotides can be quickly assessed using the fluorescent analog ATP (thrinitrophenyl-ATP, TNP-ATP). In solution, this reagent has low fluorescence; binding of TNP-ATP to proteins is accompanied by an increase in fluorescence, as shown in Fig. 10.

The specificity of TNP-ATP binding and relative affinities toward various nucleotides can then be determined by competition studies as in refs. 55-58. The disadvantage of TNP-ATP as a probe for the nucleotide-binding site is a relatively high nonspecific binding because of protein interaction with TNP moiety. However, there are also certain advantages. The TNP-ATP-based assay not only estimates the ability of the isolated domain to binds nucleotides, it also can be used to monitor the o u.

Fig. 10. The comparison of the nucleotide-binding properties for the ATP-binding domains of WNDP (filled circles) and Na+,K+-ATPase (the typical representative of the P2-type ATPases, empty circles). The identical amounts of purified nucleotide-binding domains were mixed with increasing concentrations of TNP-ATP and the nucleotide-binding was monitored by the increase in TNP-ATP fluorescence.

changes in the microenviroment of the probe. As shown in Fig. 10, whereas the affinities of ATP-BD and the ATP-binding domain of the Na pump for TNP-ATP are comparable, the increase in the TNP-ATP fluorescence is larger when it binds to ATP-BD, indicating that the microenvironment of TNP-ATP differs in ATP-BD and Na+,K+-ATPase ATP-binding domain. Therefore, one can utilize this protocol to estimate whether mutations in ATP-BD alter the surrounding environment of the nucle-otide-binding site.

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