CHAPTER 5 The Plasmodium spp. putative Cox17 copper metallochaperone
5.2 Results
5.2.4 Copper binding studies with His 6 -PyCox17 and GST-PfCox17
For the expression of His6-PyCox17 it was found that induction with 0.4 mM IPTG, for 4 hours, at 37°C was optimal, whilst for GST-PfCox17 induction with 0.5 mM IPTG, for 4 hours, at 37°C was optimal (data not shown). Each construct was then prepared for large-scale expression and purification of the recombinant protein. Following cell lysis, each recombinant protein was affinity purified using the Ni-NTA matrix for His6-PyCox17 (Figure 5.7) and glutathione-agarose for GST-PfCox17 (Figure 5.8). His6-PyCox17 yields were 3.95 mg and GST-PfCox17 6.87 mg protein per 500 ml of culture. The identity of each recombinant protein was confirmed by western blot. Purified His6-PyCox17 was detected by both an anti-His6 and anti-KTC anti- peptide antibody (Figure 5.7) and GST-PfCox17 was detected by an anti-GST and anti-NKG anti-peptide antibody (Figure 5.8). The His6-PyCox17 bands of a higher molecular weight than the monomeric species are likely to represent multimers. Although less evident, GST-PfCox17 also produced multimeric species (Figure 5.8).
Figure 5.8 Recombinant expression and affinity purification of GST-PfCox17
12.5% SDS-PAGE analysis of affinity purified GST-PfCox17 (A), detected by an anti-GST antibody (B) and anti-NKG (C) anti-peptide antibody following blotting to nitrocelulose. Lane 1, soluble cell lysate; lane 2, unbound protein fraction; lanes 3 – 5, protein eluate from glutathione-agarose affinity matrix. Molecular mass markers (M) are on the left of each gel (see materials and methods for details).
(Banci et al., 2008b). These conditions were used to emulate the reducing environment of the cell cytosol. Following overnight dialysis, protein-bound copper was detected using the copper- specific BCA release assay (Brenner and Harris, 1995). The BCA release assay was conducted on duplicate samples with or without the inclusion of ascorbic acid (Figure 5.9, solid and open bars, respectively) to identify the oxidative state of the bound copper. Similar to the previous results (Chapter 4), both His6-PyCox17 and GST-PfCox17 could be reconstituted with Cu+ in vitro as seen following protein incubation with CuCl2 in the presence of DTT and ascorbic acid (Figure 5.9a, +). The binding of the Cu+ ion, to both recombinant proteins, was supported by omitting ascorbic acid from the BCA release assay, which resulted in the formation of a detectable purple BCA-Cu+ complex (Figure 5.9a, +, open bars).
Sample CuCl2
only
His6-
PyCox17 GST-
PfCox17 GST Sample His6-
PyCox17 GST-
PfCox17 GST
Ascorbate + + + CuCl2 – + – + – +
Figure 5.9 Copper binding to His6-PyCox17 and GST-PfCox17 in vitro and in vivo
Copper binding was analysed A in vitro and B in vivo. (A) 10 µM affinity purified protein was incubated with CuCl2 in the presence (+) of ascorbic acid. The standard BCA release assay was altered to test for bound copper in the presence (solid bars) or absence (open bars) of ascorbic acid. The BCA-Cu+ complex was detected at 354 nm. The concentration of the copper standard (CuCl2 only) was equimolar to the amount of protein used. (B) 0.5 mM CuCl2
was added to the cell growth medium after induction of recombinant protein expression. Following affinity purification bound copper was detected by the BCA release assay with (solid bars) or without (open bars) the addition of ascorbic acid and the BCA-Cu+ complex detected at 354 nm. ** (for His6-PyCox17) and ▲▲ (GST-PfCox17) denote statistical significance as determined by Student's t-test (p-value <0.05). Results are means ± S.E. of triplicate measurements from each duplicate dialysis bag.
Copper binding to His6-PyCox17 and GST-PfCox17 was also evaluated in vivo. As before (Chapter 4), copper binding in vivo was determined by expressing the two recombinant proteins in the presence or absence of 0.5 mM CuCl2. Bound copper was detected using the BCA release assay. Following affinity purification and dialysis, His6-PyCox17 and GST-PfCox17 were
0 0.05 0.1 0.15 0.2 0.25
A354
A. B.
0 0.05 0.1 0.15 0.2 0.25 0.3
A354
▲▲
▲▲
**
**
shown to bind copper in vivo (Figure 5.9b), with the addition of extracellular copper producing a statistically significant increase in the amount of copper detected (p-value <0.05). The oxidation state of the bound copper was determined by including (Figure 5.9b, solid bars) or omitting (Figure 5.9b, open bars) ascorbic acid from the BCA release assay. His6-PyCox17 and GST- PfCox17 bound Cu+ in vivo (Figure 5.9b). Interestingly, when the in vitro and in vivo data are compared, a similar amount of copper was found to be bound to these two proteins under the two conditions tested (Figure 5.9). Copper did not bind to the GST carrier protein (Figure 5.9) thereby indicating that copper binding was specific to the PfCox17 partner protein.
Figure 5.10 Copper-catalysed oxidation of ascorbic acid is inhibited by His6-PyCox17 and GST-PfCox17 A 120 µM solution of ascorbic acid (H2Asc) at pH 4.5 (■). Addition of 8 µM CuCl2 to the H2Asc solution (♦). His6- PyCox17 (▼), GST-PfCox17 (▲) and GST carrier protein (►) added to the H2Asc-copper solution.
As before, the effect that the recombinant proteins had on copper-catalysed ascorbic acid oxidation was examined by monitoring the loss of absorbance, at 255 nm, of a 120 μM ascorbic acid solution at pH 4.5. This pH was selected for reasons previously described (Section 4.2.4).
As expected, the control reaction, with freshly prepared ascorbic acid, showed a persistent absorbance at 255 nm (A255) over a period of 300 seconds, indicative of the stability of the solution to aerial oxidation (Figure 5.10, ■). Upon the addition of 8 μM CuCl2, the A255 signal was found to decrease thereby highlighting the catalytic oxidation of ascorbic acid by copper (Figure 5.10, ♦). The copper-catalysed oxidation of ascorbic acid was, however, inhibited by the addition of 5 μM of purified His6-PyCox17 (Figure 5.10, ▼) or GST-PfCox17 (Figure 5.10, ▲).
The presence of either recombinant protein results in only a slight decrease in ascorbic acid stability over time, compared to the dramatic decrease seen when CuCl2 is added. This indicates that both His6-PyCox17 and GST-PfCox17 chelate copper in solution, thus preventing its participation in the redox cycle. Taken together with the previous copper binding results
0 50 100 150 200 250 300
0.4 0.6 0.8 1 1.2 1.4
Time (sec)
A255
(Figure 5.9), it is presumed that both proteins chelate the cuprous ion in solution thereby shifting the rate-limiting step to equation 2 of this redox cycle (Figure 4.23). Interestingly, for the ascorbate oxidation assay, His6-PyCox17 and GST-PfCox17 produce a very similar result (Figure 5.10). This was in contrast to the differences observed for the in vitro and in vivo copper-binding assays (Figure 5.9). Overall these studies suggest that both proteins bind Cu+ in solution. This is in agreement with findings for other Cox17 metallochaperones, such as S.
cerevisiae Cox17 (Abajian et al., 2004; Beers et al., 1997; Palumaa et al., 2004; Voronova et al., 2007b).