Oriented Attachment of Recombinant Proteins to Agarose-Coated

Nov 3, 2016 - Design of generic methods aimed at the oriented attachment of proteins at the interfacial environment of magnetic nanoparticles currentl...
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Oriented Attachment of Recombinant Proteins to Agarose-Coated Magnetic Nanoparticles by Means of a β‑Trefoil Lectin Domain Iván Acebrón,†,‡,# Amalia G. Ruiz-Estrada,§,∥,# Yurena Luengo,§ María del Puerto Morales,§ José Manuel Guisán,⊥ and José Miguel Mancheño*,† †

Departamento de Cristalografía y Biología Estructural, Instituto Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain Departamento de Biomateriales y Materiales Bioinspirados, Instituto de Ciencia de Materiales de Madrid, CSIC, Sor Juana Inés de la Cruz, 3, Cantoblanco, 28049 Madrid, Spain ⊥ Departamento de Biocatálisis, Instituto de Catálisis, CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain §

S Supporting Information *

ABSTRACT: Design of generic methods aimed at the oriented attachment of proteins at the interfacial environment of magnetic nanoparticles currently represents an active field of research. With this in mind, we have prepared and characterized agarose-coated maghemite nanoparticles to set up a platform for the attachment of recombinant proteins fused to the β-trefoil lectin domain LSL150, a small protein that combines fusion tag properties with agarose-binding capacity. Analysis of the agarose-coated nanoparticles by dynamic light scattering, Fourier transform infrared spectroscopy, and thermogravimetric studies shows that decoupling particle formation from agarose coating provides better results in terms of coating efficiency and particle size distribution. LSL150 interacts with these agarose-coated nanoparticles exclusively through the recognition of the sugars of the polymer, forming highly stable complexes, which in turn can be dissociated ad hoc with the competing sugar lactose. Characterization of the complexes formed with the fusion proteins LSL-EGFP (LSL-tagged enhanced green fluorescent protein from Aquorea victoria) and LSL-BTL2 (LSL-tagged lipase from Geobacillus thermocatenolatus) provided evidence supporting a topologically oriented binding of these molecules to the interface of the agarose-coated nanoparticles. This is consistent with the marked polarity of the β-trefoil structure where the sugar-binding sites and the N- and C-terminus ends are at opposed sides. In summary, LSL150 displays topological and functional features expected from a generic molecular adaptor for the oriented attachment of proteins at the interface of agarose-coated nanoparticles.



complex biological media, allowing imaging techniques,4,5 noncovalent conjugation enables applications in sensing2 and delivery.6,7 Many metal, semiconductor, and carbon NPs are hydrophobic once synthesized and need to be made hydrophilic and biocompatible before basic analysis or application. This usually requires the incorporation of hydrophilic ligands or coatings that favors aqueous dispersion,8 permitting the design of subsequent, more specific attachment steps of biomolecules.9 However, coating processes are chemically complex and yield rather heterogeneous systems with nonuniform NM surfaces.3 This is important since biointerfacial interactions are driven by the chemical and topological nature of NP coverage.1 Hence, it is obvious that the design of specific NM-bioconjugates faces unique challenges associated with the fundamental physical chemical processes at work at the biomolecule−NM interface.9 Obviously, a detailed characterization of these processes is

INTRODUCTION Nanoparticles (NPs) offer the possibility to prepare hybrid systems of biomolecules conjugated with nanomaterials (NMs), which have found notable applications in biotechnology, medicine, and catalysis.1 Somehow, NPs and NMs combine the fields of biology and synthetic materials.2 The versatility of the resulting NM biocomposites most probably resides in the joint capabilities emerging from this integration.3 Yet, this process faces numerous nontrivial challenges since it combines two distinct and somewhat opposing aspects: first, the intrinsic properties of biomolecules such as (stereo)specific recognition of molecules, characteristic biophysical features, or catalytic activity and, second, the unique chemical−physical properties of NMs. Hence, regardless of the particular application, the conjugation of biomolecules at the NP or NM interface becomes a crucial step. Two general approaches can be used to conjugate biomolecules to NPs or NMs: covalent linkage or noncovalent interactions.1,3 These approaches are complementary, each one with its own domain of application. Thus, whereas the first approach can provide conjugates stable toward dissociation in © XXXX American Chemical Society

Received: September 9, 2016 Revised: October 20, 2016

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DOI: 10.1021/acs.bioconjchem.6b00504 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry necessary to control key aspects such as the NM/biomolecule ratio, the orientation and distance of the biomolecule to the surface and the affinity to the surface.9 These criteria should be satisfied for an optimal performance of the NM-bioconjugates.10 Recently, we demonstrated that the interaction between the β-trefoil lectin domain of the hemolytic pore-forming toxin LSLa from the mushroom Laetiporus sulphureus (LSL150) and plain agarose beads renders highly stable complexes, despite the underlying molecular mechanism proceeding exclusively through the weak and specific recognition of the sugar moieties of the polymer.11,12 Thermodynamic analysis of lactose binding by LSL150, combined with the structural information, indicates the presence of two independent binding sites with Kd1 170 μM and Kd2 ∼11 mM. Hence, LSL150 binds lactose in solution only weakly. Most probably, the high stability of the complexes formed with the polymer is a consequence of the cluster glycoside effect, where a dramatic enhancement in affinity resulted from the multivalent nature of the ligands (avidity).13 In fact, this interaction is operatively so strong that LSL150 binding to plain Sepharose 4B has permitted the design of a generic, one-step purification protocol of LSL-tagged proteins using this matrix as affinity resin.11 Based on these results and the structural data about LSL150 that reveal a marked topological polarity between the location of the lectin sugar binding sites and that of its C-terminal end, where the accompanying protein is fused, we formed the working hypothesis that this molecule could be used as a molecular adapter for the topologically oriented attachment of proteins to the interface of agarose-coated magnetic nanoparticles (AgMNPs). Magnetic, micron-sized agarose beads are currently used in biotechnology as a platform for various biomolecule capture and purification processes. The distinguishing feature of these particles is their superparamagnetic properties, which enable their separation using external magnetic field gradients. Yet, reducing the size of these particles from 50 to 150 μm to the nanometer range will increase the available, potentially interacting surface area. Thus, in this work we have designed an efficient protocol for preparing such NPs and have characterized the interactions with LSL150 and LSL-tagged proteins.

Figure 1. Schematic representation of the procedures employed in the synthesis of agarose coated magnetic nanoparticles in one step (coprecipitation in the presence of agarose) or two steps (agarose coating after coprecipitation). The resulting particles present different aggregate sizes that are represented in the lower part of the figure according to their colloidal properties (Table 1).



RESULTS AND DISCUSSION Synthesis of Nanoparticles. Two different methods for the synthesis of iron oxide nanoparticles coated with agarose have been designed (Figure 1). Both approaches allow the production of magnetic particles coated with agarose in the nanometer range in contrast to the results obtained by other authors.14 Magnetic nanoparticles prepared by the one-step procedure (MAgr) were obtained by coprecipitation of iron salts in the presence of the polymer. The obtained particles were not well dispersed and presented a wide size range: hydrodynamic sizes of 317 nm were obtained, with PDI degrees of 0.345 (Figure 2A). Hydroxyl groups of the agarose were responsible for the low surface charge of the nanoparticles at pH 7 (−6 mV), and the isoelectric point was very close to that of uncoated maghemite nanoparticles (Figure 2B). By contrast, the particles produced by the two-step procedure were more uniform with a narrower size distribution. Mean values of hydrodynamic sizes before agarose coating were ∼50 nm for the smallest particles (M6 and M8) and ∼75 nm

Figure 2. Colloidal properties of uncoated and coated magnetic nanoparticles. (A) Z-average hydrodynamic sizes as a function of the synthesis route. (B) Evolution of Z-potential as a function of pH. Color code is as follows: red for M8, blue for MAgr, black for M6-Agr, red (dash line) for M8-Agr, and green for M12-Agr.

for M12, with PDI degrees of 0.2, and the isoelectric point was around 7, which is in agreement with previously reported values.15,16 The average hydrodynamic diameter measured after coating increased up to 256−282 nm, although the PDI degree values remained at 0.2. The data are summarized in Table 1. B

DOI: 10.1021/acs.bioconjchem.6b00504 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry Table 1. Summary of the Hydrodynamic Properties of Magnetic Nanoparticles As Synthesized (M6, M8, M12) and the Agarose-Coated Magnetic Nanoparticles Synthesized in One Step (MAgr) or Two Steps (M6-Agr, M8-Agr, M12Agr) samples

Z-average hydrodynamic size (nm)

PDI

Z-potential at pH 7 (mV)

M6 M8 M12 M6-Agr M8-Agr M12-Agr MAgr

48 55 75 256 267 282 317

0.2 0.2 0.2 0.2 0.2 0.2 0.3

15 15 15 −15 −11 −7 −6

Figure 3. SDS-PAGE analysis of the binding of LSL150 to Ag-MNPs prepared by the one-step procedure (MAgr) or the two-step procedure (M8-Agr). Binding assays were carried out as described in the Experimental Procedures section (see the text for details). LSL150 (10 μg) was incubated for 2 h at room temperature with increasing amounts of Ag-MNPs, from 10 to 60 μL of stock solutions of MAgr (2.3 mg/mL) or M8-Agr (1.3 mg/mL) in a total volume of 70 μL, respectively. 10 μL of the supernatant obtained after centrifugation of the samples at 10 000g for 5 min were then used for SDS-PAGE analysis.

The evolution of Z-potential with pH can be observed in Figure 2B. One-step agarose coating reduces the surface charge but the isoelectric point is maintained at around pH 7, which is similar than that reported for bare maghemite nanoparticles,16 indicating a poor surface modification with the polymer. However, Ag-MNPs obtained with the two-step procedure showed a significant variation in the isoelectric point from ∼7 to 2. In this case we also observed differences in the surface charge as a function of the nanoparticle size. Z-potential at pH 7 for M6-Agr is around −15 mV and −9.8 mV for M8-Agr and −7 mV for M12-Agr. This is probably due to the inverse relationship between the nanoparticle size and surface area. Binding of the β-Trefoil Lectin LSL150 to AgaroseCoated Nanoparticles. We have recently characterized the mechanism of interaction between the β-trefoil lectin domain LSL150 from the mushroom Laetiporus sulphureus and two types of agarose polymers: agarose 4B (polymerized agarose) and agarose 4BCL (polymerized agarose cross-linked with epychloridrine) 12 and also the dynamics and stability of immobilized LSL-tagged proteins.17 The binding of the lectin to agarose-based polymers is driven by lectin−sugar interactions, resulting in a strong and rapid initial binding step, followed by a highly dynamic protein distribution inside the porous beads of agarose that evolved from heterogeneous to homogeneous along the post-immobilization step. Under the hypothesis that these lectin−sugar interactions may also be the driving force for the topologically oriented attachment of lectintagged proteins to magnetic nanoparticles, we have studied the binding of LSL150 to Ag-MNPs. LSL150 bound to Ag-MNPs, prepared either by the one- or the two-step procedure (Figure 3). As can be observed, at the same nanoparticle to lectin ratio a more efficient binding was observed for the nanoparticles prepared by the two-step procedure, which is most probably due to the poorly surface modification of the MAgr nanoparticles with the polymer. Moreover, since similar results were obtained for M6-Agr, M8-Agr, and M12-Agr nanoparticles (not shown), surface charge would not play an important role in lectin binding to these nanoparticles, suggesting that recognition of the polymer may be the main factor underlying this interaction. To determine whether this is indeed the case, binding experiments were carried out with M8 nanoparticles coated with different materials: dimercaptosuccinic acid (DMSA; negatively charged at pH 7.0), aminodextran (ADX; positively charged at pH 7.0), and uncoated maghemite nanoparticles (low surface charge at pH 7.0). A 5-fold excess of nanoparticles over lectin (w/w) was used to maximize potential binding of the lectin. After 2 h

incubation at room temperature, the mixture was centrifuged (10 000g, 5 min) and the supernatant analyzed by SDS-PAGE. The results indicated that under these experimental conditions lectin binding is observed to Ag-MNPs, and unexpectedly also to uncoated maghemite nanoparticles (Figure 4A). Preincuba-

Figure 4. Binding of LSL150 to nanoparticles with different coatings and effect of lactose in the solution. Binding assays of LSL150 (10 μg) to different nanoparticles (40 μg) were done as described in the text. The total reaction volume was 50 μL. The binding experiments were done in the absence of lactose (A) and also after previous incubation of the lectin with 0.1 M lactose (B). The results obtained after addition of 0.1 M lactose to the incubation mixtures of LSL150 with the nanoparticles were indistinguishable from those shown in panel B (see Figure S1).

tion of LSL150 with lactose 0.1 M (2 h at room temperature) completely abolished lectin binding to Ag-MNPs (Figure 4B), whereas no effects are observed with DMSA- or ADX-coated nanoparticles and a minor effect is observed for uncoated nanoparticles. With the exclusion of this latter binding, which will be analyzed in more detail below, these results indicate that lectin binding is observed only to Ag-MNPs and that it is essentially driven by interactions with the sugars present in the agarose polymer of the nanoparticles. Since agarose is composed of repeating units of the disaccharide agarobiose (β-1,3-DC

DOI: 10.1021/acs.bioconjchem.6b00504 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry galactose and α-3,6-anhydro-L-galactose), the polymer contains a rich network of potential binding sites for LSL150. In fact, we have described in detail the interactions of this lectin with lactose both structurally and thermodynamically.11,18 This binding is mainly mediated by numerous weak interactions between the galactose ring of the disaccharide (the glucose unit also contributes but to a minor extent) and polar and aromatic amino acid side chains that constitute the two operative sugarbinding sites of LSL150. Key stacking interactions between the galactose ring and a conserved aromatic residue within each sugar-binding site (Tyr91 and Phe139, respectively) are identified together with a dense network of hydrogen bonds between hydroxyl groups of the sugar and the polar amino acid side chains that shape the binding sites.11 It is remarkable that, despite the great diversity of sequences and ligand-binding functions of β-trefoils,19,20 the general features of lactose binding by the family of carbohydrate-binding β-trefoil lectins are highly conserved.21 Additionally, incubation with lactose (0.1 M; 2 h at room temperature) of the reaction mixtures of LSL150 with the different nanoparticles indicated that attachment of the lectin to the agarose polymer is fully reversed by the disaccharide, whereas binding to uncoated maghemite nanoparticles is only partially reversed (Figure S1). These results reinforce the above conclusion that sugar−lectin interactions mediate lectin binding to Ag-MNPs. Moreover, since the sugar-binding sites of LSL150 are perfectly defined structurally and sugar-binding does not induce large-scale conformational changes in LSL150 nor promotes protein−protein interactions, it can be deduced that attachment of LSL150 to the Ag-MNPs is a local, interfacial process exclusively mediated by interactions between the sugarbinding sites of the lectin and the sugars of the polymer. Since, in general, biointerfacial interactions of nanoparticles are dictated by both the chemical and topological nature of the nanoparticle coverage,1 we claim that the coating processes we have designed for maghemite nanoparticles, mainly the twostep coating process, yielded properly functionalized nanoparticles for LSL150-binding since they interact exclusively through the recognition of the sugars. These results agree with previous ones indicating that interactions between properly functionalized nanoparticles and proteins share kinetic and thermodynamic characteristics with protein−protein interactions.22 As indicated previously, LSL150 unexpectedly bound to uncoated maghemite nanoparticles (Figure 4A). In contrast to the interactions with Ag-MNPs, this binding was not reversed by lactose 0.1 M after an incubation of 2 h at room temperature with a lectin to nanoparticle mass ratio of (1:10) (Figure 5A), which contrasts the reversibility observed with M8-Agr nanoparticles (Figure 5B). Therefore, binding to maghemite cores does not specifically involve the sugar-binding sites of the lectin, and most probably, considering the nature of the surface of the maghemite nanoparticles, this physical adsorption of the lectin would be based on weak hydrogen bonding interactions. The latter agrees well with our observation that prolonged incubation times (>5 h) in the presence of lactose, added either to preformed complexes M8LSL150 nanoparticles or to LSL150 before incubation with the M8 nanoparticles, notably reduce lectin binding (Figure S2). Leakage of the lectin from Ag-MNPs was not observed under identical experimental conditions (not shown). Colloidal Properties of the LSL150-Nanoparticles Complexes. To evaluate the stability and aggregation degree

Figure 5. Study of the effect of lactose on the reversibility of LSL150 binding to nanoparticles. Preformed complexes between LSL150 and M8 maghemite (A) or M8-Agr nanoparticles (B) at a lectin to nanoparticle mass ratio of (1:10) were treated with different concentrations of lactose and further incubated for 1 h at room temperature.

of nanoparticle samples after lectin binding, we determined the colloidal properties of the hydrophilic suspensions by measuring the dependence on pH of the hydrodynamic size and Z-potential. After binding of LSL150, the average hydrodynamic size of M8-Agr increased from 282 to 1563 nm at pH 7 (Figure 6A), whereas the increase for M8 uncoated

Figure 6. Characterization of the hydrodynamic size of the LSL150nanoparticles complexes consisting of uncoated (M8) and coated 8 nm magnetic nanoparticles obtained by the two-step process (M8Agr).

nanoparticles was from 42 to 82 nm (Figure 6B). Also, the net surface charge at pH 7 changed from approximately −9.8 mV for M8-Agr and 11.2 mV for M8 to −5.6 mV and −3.18 mV, respectively, for the corresponding complexes with LSL150. To properly interpret these results, we believe that three considerations are relevant: first, the overall dimensions of LSL150 are 3.5 × 3.0 × 3.0 nm3 (see PDB entries 2Y9F or 2Y9G),11 namely, LSL150 is a highly compact, globular protein domain; second, the different scale between the distance between the two operative sugar-binding sites of LSL150 (∼3 nm) and the average hydrodynamic size of the M8-Agr nanoparticles (282 nm) makes it unlikely that LSL150 acted as a bridge between two agarose-coated nanoparticles; and third, since the interaction between the lectin and the nanoparticles is based on the recognition of the sugars by the lectin, the formation of multiple layers of protein around the nanoparticle D

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carried out with M8-LSL150 (Figure 8C) and (M8-Agr)-LSL150 (Figure 8D) complexes revealed weight losses associated with the protein of ∼15% and 9%, respectively, which corresponds to 1.65 and 0.9 mg of protein. Oriented Attachment of LSL-Fusion Proteins to Agarose-Coated Nanoparticles. Since the interaction between LSL 150 and Ag-MNPs proceeds through the recognition of the sugars of the polymer, we raised the working hypothesis that LSL150 could be used as a molecular adapter for the oriented attachment of recombinant proteins to magnetic nanoparticles. We based this hypothesis on our previous results on the generic, one-step purification protocol of LSL-tagged recombinant proteins using plain Sepharose matrices,11 which in turn is allowed by the specific topological properties of the βtrefoil structure (Figure 9). The β-trefoil is a relatively small,

can be excluded. So, as a whole, we believe that these aspects indicate that the observed increase in the hydrodynamic size of the nanoparticles incubated with LSL150 should be attributed to extensive aggregation of the nanoparticles, mainly the agarosecoated ones, due to the modification of their colloidal properties upon complex formation. LSL150 binding to the agarose polymer was also studied by FTIR (Fourier transformed infrared spectroscopy). IR spectra of free M8-Agr nanoparticles and of (M8-Agr)-LSL 150 complexes are shown in Figure 7. The spectrum of M8-Agr

Figure 7. FTIR spectra of free M8-Agr nanoparticles and (M8-Agr)LSL150 complexes.

samples consists of peaks assigned to metal skeleton vibration (Fe−O) in the region of 550−600 cm−1 and a broad peak between 3000 and 3500 cm−1 due to surface −OH groups.23 Conversely, the IR spectrum for M8-Agr in complex with LSL150 revealed some characteristic peaks assignable to the presence of different amino acid side chains: 1520 cm−1 (OH of Tyr, and C−H, C−N of Trp), 1462 cm−1 (δ(CH) of Trp, C−N of Pro), 1375 cm−1 (CH3 of Val and Leu), 1150−1170 cm−1 (γCH2 of Trp and Thr), 1070 cm−1 (indole group of Trp), and 967 cm−1 (C−O Ser).24 Thermogravimetric analysis (TGA) was used to further characterize complexes between LSL150 and M8 or with M8Agr nanoparticles. First, we evaluate the coating efficiency of M8 nanoparticles with agarose by quantifying the total polymer bound. Whereas TGA of the uncoated M8 nanoparticles revealed a weight loss of ∼8% due to the removal of physical and chemical water (Figure 8A), M8-Agr experienced a total weight loss of 16% (Figure 8B), revealing that the polymer represented 8% in weight. This agrees well with the observed reduction of the surface charge after agarose conjugation to the nanoparticles (Figure 2B). Similar thermogravimetric analyses

Figure 9. Schematic representation of the β-trefoil fold of LSL150. The polypeptide backbone of LSL150 is represented as a ribbon model and the bound sugars are depicted as sticks. The marked polarity of the lectin derives from the relative locations of the sugar-binding sites and the N- and C-terminal residues, which are situated at opposite ends of the protein fold. The atomic coordinates used are those of the PDB entry 2y9g. The figure has been prepared with PyMOL.34

globular protein domain (around 150 amino acids) composed of a six-stranded antiparallel β-barrel capped at one end by three hairpin turns, which is where the sugar-binding sites are located.19 The global architecture of β-trefoils is such that the N- and C-terminal ends of the polypeptide chain and the (up to) three operative sugar-binding sites are located at opposite sides of the protein fold (Figure 9). Thus, a priori, from a topologically point of view any target protein can be expressed as a fusion construct with a β-trefoil (either N- or C-terminally)11 without interfering with their sugar-binding sites and consequently with its binding capacity to Ag-MNPs. We believe that these topological features of LSL150 together with its intrinsic agarose-binding properties12,17 make this protein an appropriate candidate as a molecular adapter for the oriented immobilization of proteins on agarosebased matrices. As expected by our previous results with agarose beads,12,17 the two model proteins herein studied, namely, LSL-EGFP and LSL-BTL2, bound to M8-Agr nanoparticles similarly to LSL150 since their binding involves the recognition of the sugar moieties of the polymer (Figure 10). In addition, both LSLtagged proteins bound to uncoated maghemite nanoparticles through a mechanism independent of the sugar-binding sites (Figure 10). Apparently, the C-terminal EGFP or BTL2 counterpart did not affect agarose binding (control experiments showed that neither EGFP nor BTL2 interacted with the nanoparticles). We have analyzed the orientation of LSL-EGFP and LSLBTL2 bound to the nanoparticles. In the first case, we used the

Figure 8. Thermogravimetric analysis of the interaction between LSL150 and M8 and M8-Agr nanoparticles. E

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in solution are indistinguishable,12 revealing not only the proper folding of the lipase upon its fusion to LSL150 but also the lack of steric interferences with the lectin, which is important since its catalytic mechanism proceeds through large conformation changes.25 This is consistent with the fact that the substrate-binding pocket of the enzyme and its N-terminal end (point of fusion with LSL150) are located at opposite sides of the molecule (PDB entry: 2W22). Measurement of the hydrolytic activity of washed complexes formed between LSL-BTL2 and Ag-MNPs revealed no loss of enzyme activity upon binding since the total specific activity observed corresponding to bound (70 ± 9 U/mL) plus unbound (180 ± 10 U/mL) LSL-BTL2 agrees well with the initial value (270 ± 10 U/mL). As would be expected from this observation, the specific activity measured for LSL-BTL2 released from the nanoparticles after lactose treatment (59 ± 1 U/mL) coincides, within the experimental error, with that from the complexes. Interestingly, these latter results are at variance with those obtained for LSL-BTL2 immobilized on agarose beads. In this case the final hydrolytic activities were 60% (bound to beads of agarose 4B) or 50% (bound to beads of agarose 4BCL) when compared to the activity measured for LSL-BTL2 released from the beads. We explained these discrepancies in terms of a rate-limiting diffusion of the substrate through the channels of the agarose beads, although the existence of a reversible inactivation of the enzyme could not be discarded.12 Now, the present results not only permit discarding enzyme inactivation upon agarose binding but the lack of a rate-limiting diffusion step for the substrate to reach the active center of the enzyme bound to the M8-Agr nanoparticles. Most probably, the agarose coverage lacks internal channels typical of agarose beads. Thus, these results point to a freely accessible active site of the lipase, which is consistent with a topologically oriented attachment of LSLBTL2 to the nanoparticles similarly to LSL-EGFP, reinforcing our former hypothesis identifying LSL150 as a molecular adapter. These results together with previous ones revealing the versatility of LSL150 as a fusion tag11,26−29 open up the possibility to use this lectin as a generic adapter for the oriented immobilization of recombinant proteins to Ag-MNPs. LSL-BTL2 also interacts with M8 nanoparticles. In fact, this binding is more efficient than that observed to M8-Agr nanoparticles since no hydrolytic activity is measured in the supernatant obtained after centrifugation (0.5 ± 0.4 U/mL). Contrary to the above results with M8-Agr, protein binding to M8 nanoparticles entailed significant enzyme inactivation since the specific activity measured (99 ± 4 U/mL) is significantly lower than the initial one (270 ± 10 U/mL). Most probably this is due to enzyme denaturation as has been described for the noncovalent binding of α-chymotrypsin conjugated with gold nanoparticles30 or the binding of transferrin to bare superparamagnetic iron oxide nanoparticles.31 In summary, these results suggest a direct contact between the lipase and the M8 nanoparticles and therefore an interaction not necessarily mediated by LSL. Practical Aspects of the Interaction between LSL150 and Agarose-Coated Nanoparticles. The interaction between LSL150 and Sepharose 4B12,17 underlie the generic, one-step affinity purification protocol of LSL-tagged proteins we reported previously.11 Now, the combination of the AgMNPs described in this work with the novel LSL-tagged TEV endoprotease (LSL*-TEV: see Experimental Procedures for details) further extends the use of this lectin in the purification

Figure 10. Binding of LSL-BTL2 and LSL-EGFP to M8-Agr and M8 nanoparticles. Binding assays were done at a protein to nanoparticle mass ratio of (1:6), with 50 μg of each fusion protein. All reaction mixtures were incubated for 2 h at room temperature. Then, lactose was added to the corresponding samples (0.1 M final concentration) and all of them were further incubated for 2 h.

accessibility of its TEV cleavage site (see Experimental Procedures for details) as a probe. A topologically oriented, local interaction mediated exclusively by LSL150 should be consistent with complete accessibility of the TEV cleavage site, and therefore complete proteolysis.11 On the contrary, a significant shielding effect against TEV digestion should be expected from a nonoriented interaction. As shown in Figure 11 TEV endoprotease cleaved all the LSL-EGFP bound to the

Figure 11. Orientation of LSL-EGFP bound to M8-Agr nanoparticles as probed by the TEV proteolysis. Binding assays were done at a protein to nanoparticle mass ratio of (1:6), with 50 μg of LSL-EGFP. After incubation of LSL-EGFP with M8-Agr nanoparticles for 2 h or overnight at room temperature, lactose was added (0.1 M total concentration) to the corresponding controls to reveal TEV digestion effect on the total protein sample (total protein). Released EGFP upon TEV digestion of bound LSL-EGFP was evident in the samples not treated with lactose (unbound protein). Incubation of the nanoparticles from the pellet after o/n incubation with lactose revealed that LSL150 remained bound to these nanoparticles after TEV digestion (+ lactose).

nanoparticles after overnight incubation: essentially, all the EGFP is released to the bulk solvent. Importantly, LSL150 remained bound to the nanoparticles after incubation with the endoprotease and is only released to the solvent upon lactose treatment. Additionally, from this latter result the absence of LSL-EGFP leakage from the nanoparticles during the incubation with TEV can be deduced. In agreement with this, a control indicated that LSL-EGFP did not dissociate from the nanoparticles in the time scale of the experiment (not shown). We believe that these results demonstrate that LSL-EGFP is topologically oriented when bound to the nanoparticles and therefore the lectin behaved as a molecular adapter. Regarding LSL-BTL2, we measured its hydrolytic activity to probe the accessibility of the active site of the lipase when bound to M8-Agr or M8 nanoparticles. Our previous studies indicated that the catalytic properties of LSL-BTL2 and BTL2 F

DOI: 10.1021/acs.bioconjchem.6b00504 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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molecular adapter permitting the attachment of proteins to agarose-coated nanoparticles. We believe that the special topology of the β-trefoil structure of LSL together with its properties as fusion tag and the specific mechanism of agarose binding are the key elements that permit defining this molecule as a generic molecular adapter. The use of magnetic nanoparticles increases the potential and practical applications of LSL150 within the recombinant protein production and purification area.

of recombinant proteins. LSL*-TEV was produced and purified as described previously for other LSL-tagged fusion proteins,11 with a final production yield of 6 mg per liter of culture. As a test case for purifying untagged recombinant proteins with LSL*-TEV and magnetic nanoparticles, we purified EGFP from LSL-EGFP. Once partially purified with Sepharose 4B affinity chromatography and further dialyzed to remove the lactose, LSL-EGFP was cleaved with LSL*-TEV as described in Experimental Procedures, producing EGFP and LSL (Figure 12). As can be observed, in this assay we have used suboptimal



EXPERIMENTAL PROCEDURES Synthesis of Iron Oxide Nanoparticles Coated with Agarose. One-Step Procedure. This method allows the preparation of iron oxide nanoparticles and their coating with agarose simultaneously. For this purpose, 100 mL of Fe(SO4)· 7H2O (10.1 g) was mixed with 10 mL of Fe(NO3)3·9H2O (8.08 g). This aqueous solution was added to 400 mL of an alkaline solution (1 M NH4OH) with 4.2 g of agarose. Nanoparticles were centrifuged (2000 rpm, 5 min) four times and the supernatant was collected. Finally the particles were centrifuged (5000 rpm, 5 min) and the fraction that remained stable in the supernatant was collected. Nanoparticles were dialyzed against distilled water for 2 days and named MAgr. Two-Step Procedure. This method consists of two main steps, a first one for the synthesis of iron oxide nanoparticles and a second one for their subsequent coating. In the first step, iron oxide nanoparticles were synthesized following the coprecipitation protocol described by Massart. 32 Some modifications, such as nature of the basis used, the addition rate, and aging time, were introduced to control particle size.15 Through a series of partial dissolution−recrystallization steps, we reduced size distribution to polydispersity degrees around 0.2 (standard deviation/mean size), and enhanced both nanoparticle colloidal and magnetic properties.33 In particular, for the preparation of 6 and 8 nm particles, 488 mL of a mixture of FeCl3·6H2O (43 mL, 27%) and 45 mL of FeCl2· 4H2O (10.8 g) were added to 75 mL of KOH (25%) and NH4OH (25%) for 6 and 8 nm, respectively. The addition was carried out at a flow rate of 40 mL·s−1, at room temperature and under vigorous stirring. The precipitate was washed three times with distilled water using a magnet (0.5 T) to collect the particles. To oxidize magnetite to maghemite (γ-Fe2O3) and activate the particle surface for further coating, the precipitate was treated with 300 mL of 2 M HNO3 under stirring for 15 min. Then, nitric acid was removed by magnetic decantation, and 75 mL of 1 M Fe(NO3)3 and 130 mL of water were added to the particles. The mixture was heated up to boiling temperature and stirred for 30 min. The particles were then cooled to room temperature and the supernatant was substituted by 300 mL of 2 M HNO3 and stirred for 15 min by magnetic separation. Finally, they were washed three times with acetone and redispersed in distilled water. A rotary evaporator was used to remove any acetone waste and concentrate the samples, which were named M6 and M8 according to the nanoparticle size. The largest particles (12 nm) were obtained by a slower addition rate (0.2 mL·s−1) over NH4OH (25%) and subjecting the nanoparticles to a heating process at 90 °C for 1 h (M12) after the coprecipitation. The remaining steps (acid treatment and washing procedure) were carried out exactly as the 6 and 8 nm samples.15 These samples were named M6, M8, and M12, respectively. The second step of the procedure, namely, the agarose coating, was carried out in a thermoregulated ultrasonic bath.

Figure 12. Purification of LSL-EGFP with the use of LSL*-TEV and M8-Agr nanoparticles. Partially purified LSL-EGFP (total LSL-EGFP, -TEV) was incubated with the new endoprotease LSL*-TEV for 4 h at room temperature at an enzyme to protein mass ratio of (1:80) (total LSL-EGFP, +TEV). Then, M8-Agr nanoparticles were added (1:6) mass ratio and further incubated for 2 h. The supernatant obtained after centrifugation at 10 000g for 5 min (S) revealed the presence of EGFP, whereas the pellet (P), upon treatment with lactose, revealed that all LSL-tagged proteins remained bound to the nanoparticles after digestion with LSL*-TEV.

conditions of proteolysis (undigested LSL-EGFP is present in the solution) to remark that all LSL-tagged species present in solution (LSL and LSL-EGFP and also the initial contaminants) would bind to the nanoparticles. The clearance produced upon incubation with M8-Agr nanoparticles yielded essentially pure, untagged EFGP (Figure 12). In summary, we believe that the system composed by LSL*-TEV and Ag-MNPs may provide a generic tool to purify LSL-tagged proteins in a straightforward manner, which would increase the potential, practical applications of LSL150 within the recombinant protein production and purification realm.



CONCLUSION We have designed two experimental approaches for the synthesis of agarose-coated maghemite nanoparticles. The one-step approach where nanoparticle formation and agarose coating are coupled processes yielded particles that were not well dispersed and exhibited hydrodynamic sizes larger than 300 nm (PDI degrees of 0.35). On the contrary, decoupling particle formation from agarose coating (two-step procedure) rendered nanoparticles more uniform with an appropriate size distribution (PDI degrees of 0.20). The β-trefoil lectin domain LSL 150 interacts with these nanoparticles through the recognition of the sugars present in the agarose polymer and therefore involves its sugar-binding sites. As expected, this interaction is fully reversible by treatment with the competing sugar lactose. The LSL-tagged fusion proteins LSL-EGFP and LSL-BTL2, whose mechanism of binding to agarose beads we have described previously, interacts with nanoparticles exclusively through LSL resulting in topologically oriented complexes. This supports our hypothesis that LSL acts as a G

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prepared in-house using glass Econo-Column columns (2.5 × 10 cm; BioRad). Prior to sample loading, the resin was washed thoroughly with binding buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 0.04% (w/v) sodium azide). Cleared cell extracts were directly loaded onto the column at 2.5 mL/min, which was then washed with binding buffer overnight at 4 °C. Fusion proteins were then eluted with elution buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.04% sodium azide (w/v), and 0.2 M lactose) at 3 mL/min. Fractions containing the eluted proteins were pooled and dialyzed overnight at 4 °C against binding buffer to remove bound lactose. Finally, a polishing size-exclusion chromatography on 16/60 HiLoad Superdex 75 (GE Healthcare) was carried out to separate the fusion proteins from potential soluble aggregates. Fractions were pooled and concentrated by ultrafiltration with YM-10 membranes (Amicon). Protein materials were stored at −80 °C. Protein purity was checked by SDS-PAGE. Regeneration of the Sepharose 4B column was done by washing with five column volumes of elution buffer and then with five additional column volumes of binding buffer. Binding Assays of LSL150 and LSL-Tagged Proteins to Agarose-Coated Magnetic Nanoparticles. The binding assays were done in Tris buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 0.04% (w/v) sodium azide) with a final volume per experiment of 50−70 μL and using 5 or 10 μg of each protein. The reaction mixtures were incubated at room temperature for 2 h and then centrifuged for 5 min at 10 000g with an Eppendorf mini Spin centrifuge. The presence of the protein in the supernatant was evaluated by SDS-PAGE. Control experiments with uncoated magnetic nanoparticles, and binding assays with nanoparticles with different coatings (dimercaptosuccinic and aminodextran) were also carried out. Considering the intrinsic complexity of nanoparticle suspensions in terms of size distribution, coating efficiency, interfacial properties, our binding assays are essentially qualitative and therefore no attempt has been made to estimate binding constants. In this sense, unless otherwise indicated binding assays have been routinely done using a low lectin to nanoparticle weight ratio (1:6 or 1:10) to maximize potential binding. To ascertain whether protein−sugar interactions underlie the binding of the lectins to the nanoparticles, the effect of lactose on these interactions was examined with two approaches: first, we studied the effect of the preincubation of the lectin with lactose (0.1 M final concentration), and second, the effect of the addition of lactose to preformed lectin−nanoparticles complexes was analyzed. With this aim, these complexes were incubated with lactose 0.1 M for 60 min at room temperature. After centrifugation of the samples, the presence of the lectin in the supernatant was estimated by SDS-PAGE as before. Molecular weight markers that appear in the gels correspond to the following masses: 200, 116, 97, 66, 45, 31, 21.5, 14.4, and 6.5 kDa. Digestion of LSL-EGFP Immobilized on AgaroseCoated Magnetic Nanoparticles with TEV Endoprotease. Agarose-coated magnetic nanoparticles (750 μg) incubated with LSL-EGFP (100 μg) for 120 min at room temperature (200 μL total volume) were washed three times with Tris buffer to remove unbound LSL-EGFP. The obtained complexes were then incubated with endoprotease TEV for 20 h at 4 °C, under gentle agitation. Aliquots withdrawn at different times were centrifuged and the supernatants were analyzed by SDS-PAGE to detect unbound protein. Bound protein, both digested and

An aqueous solution of 5 mg of nanoparticles was dispersed in 1.6 mL of 0.8 M NaOH. These nanoparticles were added very slowly (drop by drop) to agarose (5 mg) dissolved in 0.5 M NaOH (2.5 mL) (Figure 1B). The mixture was sonicated (6 h, 25 °C), followed by extensive dialysis (samples M6-Agr, M8Agr, and M12-Agr). Characterization of the Agarose-Coated Nanoparticles. Colloidal properties were characterized by dynamic light scattering (DLS) using a Nanosizer ZS (Malvern) and 0.5 mM Fe nanoparticle suspensions in water. Z-average values in intensity at pH 7 were used as mean hydrodynamic size (Dh), the size distribution was evaluated from the polydispersity index (PDI = deviation/mean size), and the Z-potential was measured in a 0.01 M KNO3 solution. HNO3 was added to the solution to change the pH. Fourier transform infrared spectroscopy (FTIR) spectra were acquired using a Nicolet 20 SXC FTIR spectrometer to confirm the iron oxide phase, the nature of the coating, and its surface bonding. IR spectra of the iron oxide nanoparticles were recorded between 250 and 4000 cm−1. Samples were prepared by diluting 2% iron oxide powder in KBr (w/w) and pressing into a pellet. Thermogravimetric analysis (TGA) of the magnetic nanoparticle powders was carried out in a Seiko TG/ATD 320 U, SSC 5200. The analysis was performed from room temperature to 1000 °C at 10 °C· min−1 with an air flow rate of 100 mL·min−1. Cloning and Production of Recombinant Proteins. The expression vectors pKLSL150, pKLSLt-EGFP, and pKLSLtBTL2 coding for the lectin LSL150 and the LSL150-tagged proteins LSL-EGFP and LSL-BTL2, respectively, were used as previously reported.11 To avoid confusion, we have simplified the nomenclature of the recombinant proteins fused to the lectin LSL150. Thus, we use the prefix LSL before the name of the protein, although in fact, the complete tag is LSLt, namely, LSL150 followed by the linker ASSS and the tobacco etch virus (TEV) endoprotease cleavage site (ENLYFQG).11 A new variant of the pKLSLt expression vector named pKLSLt* has also been prepared. In this vector, the 3′-end of the LSL150 coding sequence is followed by an in-frame sequence coding for the linker sequence ASSS, as in pKLSLt followed by a sequence coding for a seven amino acid sequence resembling TEV cleavage site (SNLSFSG) but noncleavable by TEV. This vector was used as template for the preparation of pKLSLt*-TEV, which codes for the fusion protein LSL*-TEV, namely, the endoprotease TEV N-terminally fused to the LSL150 lectin through the previously described connecting sequence. Production of the recombinant proteins was carried out as described previously.11 Briefly, the respective plasmids were used to transform E. coli BL21 (DE3) cells (Novagen, Germany). Transformed cells were grown at 37 °C in LB media containing 50 μg/mL kanamycin until the culture turbidity (OD600) reached 0.6−0.8, when they were induced with 0.3 mM isopropyl-b-D-thiogalactoside (IPTG). After induction, the cultures were grown at 16 °C for 20 h before harvesting the cells by centrifugation at 4000g for 15 min. Cell pellets were suspended in 25 mL 20 mM Tris-HCl, pH 8.0, 100 mM NaCl and flash frozen at −80 °C until further use. Purification of LSL150, LSL-EGFP, LSL-BTL2, and LSL*TEV. The purification of the recombinant proteins LSL150, LSLEGFP, LSL-BTL2, and LSL*-TEV was accomplished by affinity chromatography on Sepharose 4B at 4 °C using a BioLogic LP chromatography system (BioRad) and an Econo Gradient Pump (BioRad) as described previously.11 Columns were H

DOI: 10.1021/acs.bioconjchem.6b00504 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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ACKNOWLEDGMENTS This work was partially supported by grant No. MAT201452069-R from the Spanish Ministry of Economy and Competitiveness (M.P.M.), by C-KBBE/3293 project from the European Commission (J.M.G.) and by grant BFU201017929 from the Spanish Ministry of Science and Innovation (J.M.M.). I.A. and Y.L. hold a predoctoral FPU fellowship from the Spanish Ministry of Economy and Competitiveness. A.G.R.E. holds a predoctoral fellowship from a CSIC−CITMA collaborative project (ICMM, 2011-2014).

undigested, was estimated by incubating the corresponding complexes with lactose 0.1 M and analyzing the supernatants by SDS-PAGE as described before. Examination of the electrophoretic bands permits the estimation of the degree of the LSLEGFP digestion by the endoprotease due to the different molecular masses of LSL (18 kDa), EGFP (27 kDa), and LSLEGFP (45 kDa). Enzymatic Activity Assays of LSL-BTL2 Bound to Agarose-Coated Magnetic Nanoparticles. The complexes formed by the incubation of LSL-BTL2 (300 μg) with agarosecoated magnetic nanoparticles (750 μg) for 120 min at room temperature were washed with Tris buffer three times to remove unbound fusion protein. The lipase activity of LSLBTL2 either bound to the nanoparticles or released from them after lactose incubation of the complexes was done as described before.12,17 Obtaining Untagged EGFP with Agarose-Coated Magnetic Nanoparticles and the Novel Endoprotease LSL*-TEV. Untagged EGFP was obtained using agarose-coated magnetic nanoparticles (750 μg) and the novel endoprotease LSL*-TEV as follows: LSL-EGFP (100 μg) was incubated with LSL*-TEV for 20 h at 4 °C (200 μL total volume) at a protease:protein weight ratio of (1:80). The reaction mixture was then incubated for 2 h with agarose-coated magnetic nanoparticles (300 μg) at room temperature. A magnet was then used to separate the nanoparticles from the bulk solution. After three washing cycles with Tris buffer, the nanoparticles were resuspended with buffer containing 0.1 M lactose. SDSPAGE was used to detect the proteins present in the initial bulk solution and in the supernatant.





ABBREVIATIONS USED Ag-MNPs, agarose-coated magnetic nanoparticles; BTL2, lipase from Geobacillus thermocatenolatus; EGFP, enhanced green fluorescent protein from Aquorea victoria; LSL150, the β-trefoil lectin domain of the hemolytic pore-forming toxin LSLa from the mushroom Laetiporus sulphureus; MAgr, agarose-coated magnetic nanoparticles prepared by the one-step procedure; NMs, nanomaterials; NPs, nanoparticles



REFERENCES

(1) Rana, S., Yeh, Y-Ch., and Rotello, V. M. (2010) Engineering the nanoparticle-protein interface: applications and possibilities. Curr. Opin. Chem. Biol. 14, 828−834. (2) De, M., Ghosh, P. S., and Rotello, V. M. (2008) Applications of nanoparticles in biology. Adv. Mater. 20, 4225−4241. (3) Sapsford, K. E., Algar, W. R., Berti, L., Boeneman-Gemmill, K., Casey, B. J., Oh, E., Stewart, M. H., and Medintz, I. L. (2013) Functionalizing nanoparticles with biological molecules: developing chemistries that facilitate nanotechnology. Chem. Rev. 113, 1904− 2074. (4) Zheng, M., and Huang, X. Y. (2004) Nanoparticles comprising a mixed monolayer for specific bindings with biomolecules. J. Am. Chem. Soc. 126, 12047−12054. (5) Xu, C. J., Xu, K. M., Gu, H. W., Zhong, X. F., Guo, Z. H., Zheng, R. K., Zhang, X. X., and Xu, B. (2004) Nitrilotriacetic acid-modified magnetic nanoparticles as a general agent to bind histidine-tagged proteins. J. Am. Chem. Soc. 126, 3392−3393. (6) Allen, T. M., and Cullis, P. R. (2004) Drug delivery systems: entering the mainstream. Science 303, 1818−1822. (7) Sandhu, K. K., McIntosh, C. M., Simard, J. M., Smith, S. W., and Rotello, V. M. (2002) Gold nanoparticle-mediated transfection of mammalian cells. Bioconjugate Chem. 13, 3−6. (8) Thanh, N. T. K., and Green, l. A. W. (2010) Functionalisation of nanoparticles for biomedical applications. Nano Today 5, 213−230. (9) Algar, W. R., Prasuhn, D. E., Stewart, M. H., Jennings, T. L., Blanco-Canosa, J. B., Dawson, P. E., and Medintz, I. L. (2011) The controlled display of biomolecules on nanoparticles: a challenge suited to bioorthogonal chemistry. Bioconjugate Chem. 22, 825−858. (10) Medintz, I. (2006) Universal tools for biomolecular attachment to surfaces. Nat. Mater. 5, 842. (11) Angulo, I., Acebrón, I., de Las Rivas, B., Munoz, R., RodríguezCrespo, I., Menéndez, M., García, P., Tateno, H., Goldstein, I. J., Pérez-Agote, B., et al. (2011) High-resolution structural insights on the sugar-recognition and fusion tag properties of a versatile β-trefoil lectin domain from the mushroom Laetiporus sulphureus. Glycobiology 21, 1349−1361. (12) López-Gallego, F., Acebrón, I., Mancheno, J. M., Raja, S., Lillo, M. P., and Guisán, J. M. (2012) Directed, strong, and reversible immobilization of proteins tagged with a β-trefoil lectin domain: a simple method to immobilize biomolecules on plain agarose matrixes. Bioconjugate Chem. 23, 565−573. (13) Lundquist, J. J., and Toone, E. J. (2002) The cluster glycoside effect. Chem. Rev. 102, 555−578. (14) Safdarian, M., Hashemi, P., and Adeli, M. (2013) One-step synthesis of agarose coated magnetic nanoparticles and their

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00504. SDS-PAGE analysis of the effect of lactose on the LSL150 binding to different nanoparticles and effect of long incubation times with lactose on the binding of LSL150 to M8 nanoparticles (PDF)



Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Addresses ‡

Structural Biology and Biocomputing Programme. Spanish National Cancer Research Centre (CNIO). Melchor Fernández Almagro, 3. 28029 Madrid. Spain. ∥ School of Pharmacy. University of East Anglia. NR4 7TJ. Norwich. United Kingdom Author Contributions

I.A., A.G.R.-E., and Y.L. performed the experimental work, synthesis of nanoparticles and protein binding assays, and research studies. J.M.G. performed activity analysis of LSLBTL2. M.P.M. and J.M.M. designed research studies and wrote the manuscript. Author Contributions #

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest. I

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Bioconjugate Chemistry application in the solid phase extraction of Pd(II) using a new magnetic field agitation device. Anal. Chim. Acta 774, 44−50. (15) de la Presa, P. D. L., Luengo, Y., Multigner, M., Costo, R., Morales, M. P., Rivero, G., and Hernando, A. (2012) Study of heating efficiency as a function of concentration, size, and applied field in γFe2O3 nanoparticles. J. Phys. Chem. C 116, 25602−25610. (16) Garcell, L., Morales, M. P., Andres-Vergés, M., Tartaj, P., and Serna, C. J. (1998) Interfacial and rheological characteristics of maghemite aqueous suspensions. J. Colloid Interface Sci. 205, 470−475. (17) Orrego, A. H., García, C., Mancheño, J. M., Guisán, J. M., Lillo, M. P., and López-Gallego, F. (2016) Two-photon fluorescence anisotropy imaging to elucidate the dynamics and the stability of immobilized proteins. J. Phys. Chem. B 120, 485−491. (18) Mancheño, J. M., Tateno, H., Goldstein, I. J., Martinez-Ripoll, M., and Hermoso, J. A. (2005) Structural analysis of the Laetiporus sulphureus hemolytic pore-forming lectin in complex with sugars. J. Biol. Chem. 280, 17251−17259. (19) Murzin, A. G., Lesk, A. M., and Chothia, C. (1992) β-Trefoil fold. Patterns of structure and sequence in the Kunitz inhibitors interleukins-1 β and 1 α and fibroblast growth factors. J. Mol. Biol. 223, 531−543. (20) Ponting, C. P., and Russell, R. B. (2000) Identification of distance hologogues of fibroblast growth factors suggests a common ancestor for all β-trefoil proteins. J. Mol. Biol. 302, 1041−1047. (21) Rutenber, E., Ready, M., and Robertus, J. D. (1987) Structure and evolution of ricin B chain. Nature 326, 624−626. (22) De, M., You, C.-C., Srivastava, S., and Rotello, V. M. (2007) Biomimetic interactions of proteins with functionalized nanoparticles: a thermodynamic study. J. Am. Chem. Soc. 129, 10747−10753. (23) Roca, A. G., Marco, J. F., Morales, M. P., and Serna, C. J. (2007) Effect of nature and particle size on properties of uniform magnetite and maghemite nanoparticles. J. Phys. Chem. C 111, 18577−18584. (24) Barth, A. (2007) Infrared spectroscopy of proteins. Biochim. Biophys. Acta, Bioenerg. 1767, 1073−1101. (25) Carrasco-López, C., Godoy, C., De Las Rivas, B., FernándezLorente, G., Palomo, J. M., Guisán, J. M., Fernández-Lafuente, R., Martínez-Ripoll, M., and Hermoso, J. A. (2009) Activation of bacterial thermoalkalophilic lipases is spurred by dramatic structural rearrangements. J. Biol. Chem. 284, 4365−4372. (26) González, B., Baños-Sanz, J., Villate, M., Brearley, C., and SanzAparicio, J. (2010) Inositol 1,3,4,5,6-pentakisphosphate 2-kinase is a distant IPK member with a singular inositide binding site for axial 2OH recognition. Proc. Natl. Acad. Sci. U. S. A. 107, 9608−9613. (27) Silva-Martín, N., Molina, R., Angulo, I., Mancheño, J. M., García, P., and Hermoso, J. A. (2010) Cystallization and preliminary crystallographic analysis of the catalytic module of endolysin from Cp-7, a phage infecting Streptococcus pneumonia. Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 66, 670−673. (28) Baños-Sanz, J., Sanz-Aparicio, J., Whitfield, H., Hamilton, C., Brearley, C. A., and González, B. (2012) Conformational changes in inositol 1,3,4,5,6-pentakisphosphate 2-kinase upon substrate binding. J. Biol. Chem. 287, 29237−29249. (29) Franco-Echevarria, E., Baños-Sanz, J. I., Monterroso, B., Round, A., Sanz-Aparicio, J., and González, B. (2014) A new calmodulin binding motif for inositol 1,4,5-trisphosphate 3-kinase regulation. Biochem. J. 463, 319−328. (30) You, C.-C., De, M., and Rotello, V. M. (2005) Contrasting effects of exterior and interior hydrophobic moieties in the complexation of aminoacid-functionalized gold clusters with αchymotrypsin. Org. Lett. 7, 5685−5687. (31) Mahmoudi, M., Shokrgozar, M. A., Sardari, S., Moghadam, M. K., Vali, H., Laurent, S., and Stroeve, P. (2011) Irreversible changes in protein conformation due to interaction with superparamagnetic iron oxide nanoparticles. Nanoscale 3, 1127−1138. (32) Massart, R. (1981) Preparation of aqueous magnetic liquids in alkaline and acidic media. IEEE Trans. Magn. 17 (2), 1247−1248. (33) Costo, R., Bello, V., Robic, C., Port, M., Marco, J. F., Morales, M. P., and Veintemillas-Verdaguer, S. (2012) Ultrasmall iron oxide

nanoparticles for biomedical applications: Improving the colloidal and magnetic properties. Langmuir 28, 178−185. (34) DeLano, W. PyMOL, http://www.pymol.org.

J

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