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Bioconjugate Chem. 2008, 19, 1964–1967
Nitrilotriacetic Acid-Derivatized Quantum Dots for Simple Purification and Site-Selective Fluorescent Labeling of Active Proteins in a Single Step ´ ngeles Touceda-Varela, Dominic J. Campopiano, and Juan C. Mareque-Rivas* Manish Gupta, Anne Caniard, A Received July 3, 2008; Revised Manuscript Received August 16, 2008
We demonstrate that QDs coated with nitrilotriacetic acid (NTA) bound to Ni2+ can be used to reversibly and selectively bind, purify, and fluorescently label His6-tagged (N-terminal) glutathione S-transferase (GST) in one step with retention of enzymatic activity. We find binding to be less effective in the absence of the His6-tag or Ni2+ ions.
Colloidal semiconductor nanocrystals (quantum dots, QDs) have emerged as powerful fluorescent probes for biological imaging applications (1-5). QDs have several advantages over small organic dyes and fluorescent proteins such as size-tuneable photoluminescence, wide excitation-narrow emission properties, improved brightness, and high resistance to photobleaching and degradation. To conjugate QDs to biomolecules, the QD surface is derivatized in such a way as to allow the attachment of the biomolecule through a covalent bond, electrostatic, or hydrophobic interactions. A typical biomolecule contains many residues capable of forming covalent and noncovalent linkages with the QD. The ability to control the site of attachment is important to ensure that the biomolecule bound to the QD is still active. Site-specific noncovalent binding of QDs to biomolecules has been achieved by exploiting carbohydrate-lectin and streptavidin-biotin interactions (6, 7). A common approach to facilitate protein purification involves the use of genetically encoded oligohistidine (Hisn) tags (8-10). Since Hisn-tags are recognized by nitrilotriacetic acid (NTA) complexes of nickel(II) (Scheme 1), purification of these proteins is achieved by passing the protein mixture through chromatography columns containing Ni-NTA resins (11-13). Given the widespread use of Hisn-tags and the fact that they can be introduced into regions of peptides where they do not disturb protein structure and function such as at the termini or in loops, they provide a convenient way to achieve site-specific binding for many applications (14-18). Hence, the fluorescent labeling of specific proteins by encoding Hisn-tags on them coupled to the recognition of these tags by Ni-NTA has become popular. Initially, this was done using organic dye-NTA conjugates (19, 20), but very recently it has been extended to QDs. Thus, during the final stages of preparation of this manuscript the first study showing that Ni-NTA-containing QDs that can be applied to imaging His6-tagged proteins in live cells has appeared (21). This is interesting because it has been shown that Hisn-tags can also bind with very high affinity (Kd ≈ 1 nM) to QDs with carboxylic acid functionalities requiring fewer synthetic steps and cheaper reagents (22). In another study, however, it was reported that QDs with carboxylic acids presented at the surface bind His-tagged proteins only in the presence of Ni2+ cations (23). Thus, it is interesting to investigate the advantages, disadvantages and applicability of each of these functionalities for the formation of QD-protein conjugates. Here, we investigate binding of His6-tagged and untagged GST as model protein to CdSe-ZnS core-shell nanoparticles with carboxylates, NTA, and Ni2+-bound NTA at the surface. * Corresponding author. E-mail:
[email protected].
Scheme 1a
a (A) Reversible binding between a His-tagged protein and Ni(NTA). (B) Synthesis of the QD functionalized with NTA.
QDs-GST conjugates are interesting because GSTs catalyze the nucleophilic addition of GSH to the electrophilic center of a range of nonpolar substrates as a way of detoxifying a wide range of harmful endogenous and xenobiotic compounds (24), and in drug resistance mechanisms (25). Since GST activity requires not only the formation of a dimeric structure (i.e., protein-protein interactions) but also binding of both GSH and an acceptor substrate (i.e., protein-substrate interactions), it is a good enzyme to investigate the effect of specific and nonspecific binding of QDs on enzymatic activity. We report that the combination of His6-tagged GST with CdSe-ZnS core-shell nanoparticles coated with Ni2+-bound NTA gives the best results. The resulting QD-protein conjugate is strongly fluorescent and readily purified by filtration or ultracentrifugation, which should enable widespread use of these QDs as a “two-in-one” purification-fluorescence labeling tool. Moreover, the QD binds the His6-tagged GST with little disruption of its enzymatic activity, whereas QD binding is weaker and disrupts enzymatic activity when GST lacks a His-tag or when Ni2+ is unavailable. This is a significant result in that it shows that by using this simple construct protein purification, fluorescent tagging, and precise positioning of the fluorescent probe to preserve structural/functional properties are all accomplished in a single, inexpensive step. It shows also that QDs presenting Ni-NTA at the surface provide a good alternative to sitespecifically binding His-tagged proteins if, like in this case, the easier-to-make QDs with carboxylic acids at the surface fail to do so (Vide infra). The QDs decorated with NTA were easily prepared from the reaction of CdSe-ZnS core-shell dihydrolipoic acid (DHLA)-
10.1021/bc800273j CCC: $40.75 2008 American Chemical Society Published on Web 09/19/2008
Communications
Figure 1. Photoluminescence spectra for CdSe-ZnS core-shell QDs coated with DHLA-NTA and DHLA-Ni(NTA) (spectra were acquired in 20 mM PBS pH 6.7, excitation at 350 nm; T ) 293 K). The integrated emission decreases 15% upon Ni2+ addition.
capped QDs (26) with commercially available N,N-bis(carboxymethyl)-L-lysine hydrate using EDC and N-hydroxysuccinimide as coupling agents in phosphate buffer solution (Scheme 1). The product is purified by filtration with a Nanosep 100K centrifugal device (Pall Corporation). The composition of the QDs was examined by X-ray photoelectron spectroscopy (XPS). The XPS spectra showed the main diagnostic peaks of the product QDs: a 2s peak at 400.1 eV due to N, and 2p3 and 2p1 peaks at 857.1 and 874.1 eV, respectively, due to Ni2+ (Supporting Information). The photoluminescence intensity of the Ni-NTA-capped QD is ca. 85% that of the NTA-capped QD (Figure 1). This is important because the paramagnetic Ni2+ was found to strongly quench the photoluminescence of some organic dyes, limiting their applications (19). We selected GST from the helminth worm Schistosoma japonica (SjGST, 26 kDa monomer) as our target enzyme because it is amenable to recombinant overexpression in E. coli as a His6-tagged construct and has been well-characterized (9). It is important to note that the His6-tag was genetically fused at the N-terminus. The noncovalent attachment of His6-tagged and untagged GST to the QD surface before and after derivatization with Ni-NTA was analyzed by SDS-PAGE. A solution of the QD or PBS (as control) was incubated with the corresponding enzyme for 2 h and passed through a Nanosep 300K centrifugal device. The retenate was redissolved in PBS buffer, and both retenate and filtrate were analyzed by SDS-PAGE. In the absence of the QD, His6-tagged and untagged GST were found only in the filtrate. Several reports have shown that the DHLAcoated CdSe-ZnS core-shell QDs are capable of binding Histagged proteins by coordination to Zn2+ ions at the nanocrystal surface (22, 26-28). Our SDS-PAGE studies, however, did not find protein in the retenate (Figure 2). Lack of binding could be due to steric hindrance at the N-terminus location of the His6tag preventing access to the Zn2+ atoms of the nanoparticle. It is also possible that the different synthetic procedures used to prepare QDs lead to subtle changes at the QD surface which affect binding of biomolecules. However, it is worth noting that it is not rare for His-tagged proteins to exhibit different properties depending on whether the His-tag is at the N- or C-terminus, and that in the studies reporting direct His-tag binding to carboxylate-coated QDs the His-tag was located at the C-terminus (22, 26-28). In contrast, using the same experimental conditions the Ni-NTA-capped QDs immobilized both enzymes. Moreover, we found more His6-tagged than untagged GST in the retenate. Binding was also investigated in the presence of high salt concentrations. His6-GST binding to Ni-NTA-capped QDs was not affected by 1 M NaCl. In contrast, untagged GST did not bind to the QDs under these conditions,
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Figure 2. SDS-PAGE of the retenate (R) and filtrate (F) after ultrafiltration through a Nanosep 300K filter of His6-GST (a), untagged GST (b), His6-GST incubated with QD (c), and untagged GST incubated with QD (d). In each case, the enzyme and QD concentrations were 16.5 µM and 9.0 µM, respectively.
Figure 3. (A) SDS-PAGE studies of the cell lysate containing His6tagged GST (lane 2) and proteins released from the Ni-NTA-coated QDs treated with PBS containing 0.5 M imidazole (lane 3) and supernatant (lane 4) after ultracentrifugation. Lane 1 is the molecular weight marker. (B) Images of the cell lysate after ultracentrifugation and of the pure QD-bound His6-tagged GST.
which suggests it is predominantly electrostatic. Thus, high salt concentrations can be used to avoid binding of untagged proteins while ensuring binding of the desired His6-tagged target. The enzyme was easily released from the QD surface upon addition of 0.5 M imidazole, which competes for the Ni2+ binding sites. Thus, decorating the surface of the QD with Ni2+ complexes of NTA seems a good approach for noncovalent site-specific fluorescent labeling of proteins, which can be used for instance if carboxylate-functionalized QDs lacking Ni2+ ions fail. Potential advantages of attaching Ni-NTA units to QDs could be stronger interactions with the His-tag (Kd ≈ 10-13 M) (18) and less sensitivity to steric hindrance and surface properties by being further away from the nanocrystal surface. Recently, the value of magnetic nanoparticles as affinity probes to selectively trap and separate His-tagged proteins from cell lysates has been elegantly demonstrated (29-32). The protein purification efficiency of the Ni-NTA-capped QDs was investigated by incubating cell lysates containing His6-tagged GST for 2 h. Remarkably, pure fluorescently labeled GST was obtained simply by ultracentrifugaton of this mixture (Figure 3). Thus, by using the Ni-NTA-capped QDs it is possible to purify and fluorescently label His-tagged proteins in a single step. Current methods for efficiently purifying and fluorescently labeling His-tagged proteins need various labor-intensive and expensive steps, such as conjugation of NTA derivatives on support materials or the preparation of suitable magnetic nanoparticles for purification purposes, followed by the attachment of fluorescent tags. Another construct suitable for onestep protein purification and site-specific labeling was recently developed and involves organic fluorophore-doped Ni-NTAmodified silica nanoparticles (33). In order to obtain information about the effect of QD binding on the catalytic activity of GST, we have used 1-chloro-2,4dinitrobenzene (CDNB) as substrate. The GST-catalyzed reaction of GSH with CDNB produces a dinitrophenyl thioether
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ACKNOWLEDGMENT We are grateful to EaStCHEM for a PhD studentship to M.G. A.T.V. acknowledges the Xunta de Galicia (Spain) for a postdoctoral fellowship. A.C. is funded by a Marie Curie Fellowship. We acknowledge support from the EPSRC to purchase the XPS. We would like to thank Dr. Wuzong Zhou and Ross Blackley at EaStCHEM-St. Andrews for the HRTEM studies. Supporting Information Available: Details on experimental procedures and characterization data. This material is available free of charge via the Internet at http://pubs.acs.org.
LITERATURE CITED
Figure 4. (A) Activity of His6-tagged (N-terminal) and untagged GST in the presence of Ni-NTA coated QDs. Conditions: [GSH] ) 8 mM, [CDNB] ) 2 mM in PBS (pH 6.7), T ) 298 K. The QD alone did not have any activity. (B) X-ray crystal structure of the SjGST homodimer highlighting the catalytically crucial Tyr7 residue in red, and the N-terminus site for the His6-tag in green (left); surface charge distributions (right).
which can be conveniently detected spectrophotometrically at 340 nm (34). His6-tagged GST and untagged GST were incubated with the same concentration of Ni-NTA-capped QD. We have found that His6-tagged GST retains its activity after binding to the QD, whereas the untagged GST loses activity (Figure 4). We suggest that the ability of the His6-tag to control the position of the Ni-NTA-capped QD relative to the GST active site is responsible for preserving the activity of the enzyme. The X-ray crystal structure of SjGST (35) shows that the N terminus, which is where the His6 tag was placed, is ca. 25 Å away from the essential catalytic residue Tyr7 (Figure 4). We have examined the distribution of positively and negatively charged residues and found that there are positive and negative regions close to the active site. These are sites where in the absence of the His6 tag nonspecific electrostatic binding could occur, disrupting the enzyme activity. By comparing the activity of the enzyme which did not bind to the QD with that of the enzyme before incubation with QD, we estimated the protein binding capacity and number of His6-GST molecules immobilized on each QD (∼16). This surface coverage correlates well with that found for QDs and proteins of similar size (26, 27). In summary, we have shown that Ni-NTA-coated QDs provide a straightforward method to, in one step, purify and fluorescently reversibly label proteins. By using these QDs, we have selectively purified and labeled an N-terminal His6-tagged GST, which was not possible using QDs with carboxylates at the surface. Moreover, we have found that Ni2+ provides a docking site which helps to precisely orient the fluorescent nanoparticle on the protein surface and that, as a result, GST retained its activity. The use of His-tags has been broadly adopted in the molecular biology and biochemistry communities, and therefore this specific conjugation strategy should enable widespread use of these QDs for a broad range of biological applications.
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