CHAPTER 4 The Plasmodium spp. putative copper transport protein: Native protein
4.2 Results
4.2.4 Copper binding studies with MBP-PbCtrNt, MBP-PfCtr211Nt TD and
The MBP-PbCtrNt aggregate was tested for its ability to bind copper in vitro using 10 µM purified recombinant protein incubated with a 20-fold molar excess of copper in the presence or absence of the reducing agent ascorbic acid. Ascorbic acid was added to determine whether the amino terminal domain of the P. berghei copper transporter preferentially bound the cuprous (Cu+) or cupric (Cu2+) ion. Following overnight dialysis, bound copper was detected using the copper-specific BCA release assay (Brenner and Harris, 1995). BCA is only capable of forming a detectable purple complex when bound to the cuprous ion, therefore ascorbic acid is included in the assay to ensure copper ion detection (Figure 4.19a) (Brenner and Harris, 1995). This property was utilised to help establish the copper-binding preference of MBP- PbCtrNt. Following copper binding and overnight dialysis, the BCA release assay was conducted with or without a second treatment of ascorbic acid (Figure 4.19b). Thus if ascorbic acid was required for the formation of a purple BCA complex it suggests that Cu2+ is preferentially bound. Alternatively, if ascorbic acid supplementation was not required for colour formation then it suggests Cu+ is the preferred substrate. With this in mind copper binding to MBP-PbCtrNt was analysed.
Figure 4.19 Recombinant protein copper binding studies using the BCA release assay for copper detection A. Standard protocol for the BCA assay, used for the detection of copper in solution. B. In vitro copper binding to recombinant protein, in the presence or absence of ascorbic acid using a modified BCA assay to determine the oxidation state of protein-bound copper.
+Ascorbate
−Ascorbate +CuCl2 +CuCl2
Dialyse
Dialyse
BCA assay
+Ascorbate
−Ascorbate protein
OR
OR Is Cu bound?
Cu+
or Cu2+
?
No Yes
Cu2+ Cu+
12 000 g Cu
Cu Cu
Cu
+TCA
OR
negative
no Cu in solution positive Cu in solution 2 min at RT
Cu Cu Cu
Cu Cu
+Buffered BCA +Ascorbate*
A.
B.
Results from the in vitro copper-binding experiments indicated that MBP-PbCtrNt had a binding preference for Cu+ since copper binding appeared to be dependent on the addition of ascorbic acid during incubation with free CuCl2 (Figure 4.20a, +). This was supported by omitting ascorbic acid from the BCA release assay, which still resulted in the formation of the purple BCA-Cu+ complex (Figure 4.20a, +, open bar). The exclusion of ascorbic acid from the initial CuCl2 incubation step resulted in a 76% reduction in the amount of BCA-Cu+ complex formed (Figure 4.20a, –), which was statistically significant according to Student's t-test (p-value
<0.05). These in vitro studies were subsequently followed up with an in vivo copper binding study. Recombinant MBP-PbCtrNt was expressed in the presence or absence of 0.5 mM CuCl2, which is a concentration of copper salt that does not affect E. coli cell growth (Lutsenko et al., 1997). Following amylose purification, MBP-PbCtrNt was analysed for binding of copper using the BCA release assay, as described above. As seen in figure 4.20b, copper binds MBP- PbCtrNt in vivo with the detection of bound copper appearing dependent upon the addition of extracellular copper. Adding extracellular copper resulted in a statistically significant increase in the amount of copper bound to MBP-PbCtrNt, as determined by Student's t-test (p-value
<0.05). As with the in vitro analysis, the oxidation state of the bound copper was determined by either including (solid bars, Figure 4.20b) or excluding (open bars, Figure 4.20b) ascorbic acid from the BCA release assay. As with copper binding in vitro, the preferred oxidation state of in vivo bound copper appears to be Cu+ (Figure 4.17). Importantly, in the control experiments for both in vivo and in vitro conditions, copper did not show significant levels of binding to the MBP carrier protein (Figure 4.20). These results suggest the amino terminal domain of the P. berghei copper transport protein binds copper. Although MBP-PbCtrNt appears to be predominantly aggregated, it still appears to possess a native-like fold allowing for metal coordination.
Sample CuCl2
only
MBP-PbCtrNt MBP Sample MBP-PbCtrNt MBP
Ascorbate – + – + CuCl2 – + – +
Figure 4.20 Copper binding to MBP-PbCtrNt in vitro and in vivo
A 10 µM affinity-purified MBP-PbCtrNt or MBP was incubated with CuCl2 in the presence (+) or absence (–) of ascorbic acid in vitro. The BCA release assay was used to detect copper with (solid bars) or without (open bars) the addition 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 the induction of recombinant protein expression. Following affinity purification, copper bound in vivo was detected by the BCA release assay with (solid bars) or without (open bars) the addition of ascorbic acid. As before the BCA-Cu+ complex was detected at 354 nm. ** denotes 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.
For in vitro copper binding studies with MBP-PfCtr211NtTD and MBP-PfCtr369NtTD, the conditions used were the same as those used for MBP-PbCtrNt. Protein-bound copper was again detected using the copper-specific BCA release assay (Brenner and Harris, 1995), with or without a second treatment of ascorbic acid following copper binding and overnight dialysis (Figure 4.19b). The data indicated that both MBP-PfCtr211NtTD and MBP-PfCtr369NtTD bound copper in vitro with an apparent preference for Cu+ (Figure 4.21a). The exclusion of ascorbic acid from the initial CuCl2 incubation step resulted in relatively low levels of bound copper for both recombinant proteins (Figure 4.21a, –). On the other hand, the inclusion of ascorbic acid to the CuCl2 incubation step resulted in an ~80% increase of copper levels bound to MBP- PfCtr211NtTD and ~70% increase for MBP-PfCtr369NtTD. Both these increases were found to be statistically significant, as determined by Student's t-test (p-value <0.05). The omission of ascorbic acid from the BCA release assay also supports a preference for Cu+, since this still results in the formation of the purple BCA-Cu+ complex for both proteins (Figure 4.21a, +, open bar).
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2
A354
A. B.
**
**
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18
A354
**
**
Sample CuCl2
only
MBP- PfCtr211NtTD
MBP-
PfCtr369NtTD MBP Sample MBP-
PfCtr211NtTD
MBP-
PfCtr369NtTD MBP
Ascorbate – + – + – + CuCl2 – + – + – +
Figure 4.21 Copper binding to MBP-PfCtr211NtTD and MBP-PfCtr369NtTD in vitro and in vivo
A 10 µM of affinity-purified MBP-PfCtr211NtTD, MBP-PfCtr369NtTD or MBP was incubated with CuCl2 in the presence (+) or absence (–) of ascorbic acid in vitro. The BCA release assay was used to detect copper with (solid bars) or without (open bars) the addition 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 the induction of recombinant protein expression. Following affinity purification, copper bound in vivo was detected by the BCA release assay with (solid bars) or without (open bars) the addition of ascorbic acid. As before the BCA-Cu+ complex was detected at 354 nm. ** (for MBP-PfCtr211NtTD) and ▲▲ (for MBP- PfCtr369NtTD)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.
Following E. coli growth and protein expression, in the presence of 0.5 mM CuCl2, MBP- PfCtr211NtTD and MBP-PfCtr369NtTD were subsequently amylose purified and analysed for the presence of copper. Both recombinant proteins were found to bind copper in vivo, with detectable copper levels dependent on the addition of extracellular copper (Figure 4.21b). The increase in the amount of copper bound to MBP-PfCtr211NtTD and MBP-PfCtr369NtTD in vivo when extracellular copper was added, was found to be statistically significant (p-value <0.05).
As with the in vitro analysis, the oxidation state of the bound copper was determined by including (Figure 4.21b, grey bars) or omitting (Figure 4.21b, open bars) ascorbic acid from the BCA release assay. For both MBP-PfCtr211NtTD and MBP-PfCtr369NtTD Cu+ appeared to be bound in vivo; however, it appeared to form a smaller proportion of the total bound copper, particularly for MBP-PfCtr211NtTD. This result did, however, indicate a similarity between copper binding in vitro and in vivo. As before, control experiments for the in vivo and in vitro conditions indicated copper did not bind to the carrier protein MBP (Figure 4.21).
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18
A354
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16
A354
A. B.
**
**
▲▲
▲▲
**
▲▲
**
▲▲
MBP MBP-
PfCtr211NtTD MBP- PfCtr369NtTD
Figure 4.22 MBP-PfCtr211NtTD and MBP-PfCtr369NtTD protein yields
E. coli host cells were grown in the absence (open bars) or presence (solid bars) of extracellular CuCl2 and the yields of affinity purified recombinant protein determined. The MBP carrier protein alone was included as a control. ** (for MBP-PfCtr211NtTD) and ▲▲ (for MBP-PfCtr369NtTD) denote statistical significance, as determined by Student's t-test (p-value <0.05).
Analysis of copper binding in vivo established the ability of the amino terminal domain of each of the P. falciparum copper transport proteins to bind copper in a cellular environment. As previously mentioned, the detection of a significant concentration of bound copper appeared dependent on the presence of extracellular copper. A consequence of adding extracellular copper to the E. coli growth medium was a statistically significant increase (p-value <0.05) in the yield of both recombinant P. falciparum proteins (Figure 4.22). A similar phenomenon was not observed for the cytoplasmically expressed P. berghei protein (data not shown). Under standard conditions the P. falciparum recombinant protein yields were found to be poor, particularly for MBP-PfCtr211NtTD with an average yield of 0.74 mg per litre of culture, whilst MBP-PfCtr369NtTD had a slightly improved yield of 1.53 mg per litre of culture. However, expression in the presence of 0.5 mM CuCl2 significantly improved these yields with an approximate 5-fold increase for MBP-PfCtr211NtTD to 3.76 mg per litre of culture and a 4-fold increase for MBP-PfCtr369NtTD to 5.98 mg per litre of culture(Figure 4.22). When expressed under the same culture conditions, a comparative analysis for MBP alone indicated minimal alteration to protein yield when CuCl2 was added to the growth medium (Figure 4.22).
Previously it has been demonstrated that peptides representative of the copper transport protein's methionine motifs bind Cu+ with methionine-only coordination (Jiang et al., 2005). In that study, the oxidation rate of ascorbic acid was used to demonstrate the ability of the
0 1 2 3 4 5 6 7
Protein yield (mg)
**
▲▲
methionine peptides to bind Cu+. Ascorbic acid is stable to aerial oxidation in the absence of metal ions, but the presence of iron or copper ions catalyse its oxidative degradation via equations 1 and 2 (Figure 4.23) (Jiang et al., 2005). Oxidative degradation results in a loss of ascorbate's characteristic, pH-dependent absorption band between 245 and 265 nm (Buettner, 1988). This is therefore a convenient spectroscopic signal for the presence of catalytic metal ions in solution. The pH-dependent oxidation rate of the reaction is first-order, with respect to both the ascorbate monoanion (HAsc—) and the metal ion. The oxidation rate is, however, known to decrease in the presence of metal chelators with slower oxidation rates indicative of more stable chelates (Khan and Martell, 1967). Chelators that favour Cu2+ slow reaction rates by making equation 1 rate-limiting, whilst chelators that stabilise Cu+ shift the rate-limiting step to equation 2 (Figure 4.23). This effect was previously demonstrated for the copper-catalysed oxidation of ascorbate in aqueous acetonitrile (Mi and Zuberbühler, 1992). Similarly the spectroscopic signal, provided by the ascorbate UV-vis assay, was used to demonstrate the ability of methionine peptides to bind Cu+ (Jiang et al., 2005). For the current study, the ascorbate UV-vis assay was used to analyse the ability of MBP-PfCtr211NtTD and MBP- PfCtr369NtTD to bind copper in vitro. This assay was also employed to gain further insight into the preferred oxidation state of bound copper.
Equation 1: HAsc— + Cu2+ → HAsc•— + Cu+ Equation 2: Cu+ → Cu2+
Figure 4.23 Equations representing the copper-catalysed oxidative degradation of ascorbic acid
The effect 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 particular pH was selected for several reasons. First, the oxidation rate of ascorbic acid increases with increased concentration of HAsc—, which has a pKa of 4.1, therefore using elevated pH values can amplify unwanted errors. Second, the hydrolysis of Cu2+
is minimal at this lower pH (Jiang et al., 2005). Last and perhaps most important, hCtr1- transfected human cells show increased copper uptake at a low extracellular pH (Lee et al., 2002). As expected, the freshly prepared ascorbic acid control showed a constant absorbance at 255 nm (A255) over a period of 300 seconds, indicative of the stability of the solution (Figure 4.24, ■). When 8 μM CuCl2 was added, the A255 signal was found to decrease highlighting the catalytic oxidation of ascorbic acid by copper (Figure 4.24, ▼). The oxidation of ascorbic acid was, however, inhibited by the addition of 5 μM purified MBP-PfCtr211NtTD (Figure 4.24, ▲) or MBP-PfCtr369NtTD (Figure 4.24, ►). The presence of either recombinant protein results in only a slight decrease in ascorbic acid stability over time, compared to the dramatic decrease when
O2
CuCl2 was added. This result indicates that both MBP-PfCtr211NtTD and MBP-PfCtr369NtTD chelate copper in solution, thus preventing its participation in the redox cycle (Figure 4.23).
Although this assay is not definitive of the copper species being preferentially bound by each protein, it is likely to be Cu+ based on the in vitro and in vivo copper-binding studies (Figure 4.21). Thus it is highly likely that equation 2 is the rate-limiting step of the redox cycle (Figure 4.23).
Figure 4.24 Copper-catalysed oxidative degradation of ascorbic acid in the presence of MBP-PfCtr211NtTD and MBP-PfCtr369NtTD
120 µM ascorbic acid (H2Asc) at pH 4.5 (■). Addition of 8 µM CuCl2 to the H2Asc solution (▼). MBP-PfCtr211NtTD (▲), MBP-PfCtr369NtTD (►) or MBP (♦) were added to the CuCl2/H2Asc solution.