Affinity-Driven Immobilization of Proteins to Hematite Nanoparticles

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Affinity-Driven Immobilization of Proteins to Hematite Nanoparticles Elaheh Zare-Eelanjegh, Debajeet K Bora, Patrick Rupper, Krisztina Schrantz, Linda Thöny-Meyer, Katharina Maniura-Weber, Michael Richter, and Greta Faccio ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03284 • Publication Date (Web): 18 Jul 2016 Downloaded from http://pubs.acs.org on July 22, 2016

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ACS Applied Materials & Interfaces

Affinity-Driven Immobilization of Proteins to Hematite Nanoparticles Elaheh Zare-Eelanjegh†, Debajeet K. Bora‡, Patrick Rupperᶲ, Krisztina Schrantz‡, Linda ThönyMeyer†, Katharina Maniura-Weber†, Michael Richter†, Greta Faccio†* †Laboratory for Biointerfaces, Empa, Swiss Federal Laboratories for Materials Science and Technology, Lerchenfeldstrasse 5, 9014 St. Gallen (Switzerland). ‡Laboratory for High Performance Ceramics, Empa, Swiss Federal Laboratories for Materials Science and Technology, Überlandstrasse 129, 8600 Dübendorf (Switzerland). ᶲLaboratory for Advanced Fibers, Empa, Swiss Federal Laboratories for Materials Science and Technology, Lerchenfeldstrasse 5, 9014 St. Gallen (Switzerland). KEYWORDS: hematite nanoparticle functionalization, protein immobilization, C-phycocyanin, laccase activity, immobilization enhances activity, surface functionalization.

ABSTRACT Functional nanoparticles are valuable materials for energy production, bioelectronics, and diagnostic devices. The combination of biomolecules with nano-sized material produces a new hybrid material with properties that can exceed the ones of the single components. Hematite is a widely available material that has found application in various sectors such as in sensing and solar energy production. We report a single-step immobilization process based on affinity and achieved by genetically engineering the protein of interest to carry a hematite-binding peptide. Fabricated hematite nanoparticles were then investigated for the immobilization of the two biomolecules C-phycocyanin (CPC) and laccase from Bacillus pumilus (LACC) under mild conditions. Genetic engineering of biomolecules with a hematite-affinity peptide led to a higher extent of protein immobilization and enhanced the catalytic activity of the enzyme.

and stability6, 7. This phenomenon can be explained by a combined contribution of the increased enzyme density, the positive contribution of the NP morphology and of the NP surface chemistry on the protein conformation, and a more favorable enzyme orientation towards the substrate6. The most common immobilization method is based on the use of chemical cross-linkers such as glutaraldehyde. Its reaction is however difficult to control involving the numerous amino groups exposed on the surface of the biomolecule, and often leading to high loading of the biomolecules on the solid carrier but with a compromised functionality8. Alternatively, the immobilization in a site-specific manner ideally provides the controlled anchoring of the biomolecule to the surface, preservation of the catalytic activity of the biomolecule, and it can be carried out for example by Staudinger ligation9, by inserting unnatural amino acids10, by ‘click’ chemistry, by chemo-enzymatic or enzymatic methods11, 12, or by protein trans-splicing13.

INTRODUCTION The controlled immobilization of biomolecules such as proteins and enzymes onto an inorganic conductive substrate is the prerequisite for the development of biocatalysts, biosensors, analytical devices, and bio-hybrid solar cells. Wide availability, nontoxicity, and biocompatibility make iron oxide materials suitable for applications in sectors ranging from energy production1 to MedTech2. Hematite (α-Fe2O3) is an antiferromagnetic material that has been applied to sensors3 and solar energy production1. The decoration of materials with proteins can be performed in many ways from adsorption that is the most direct but also the most uncontrollable process, to covalent immobilization using chemical or enzymatic crosslinkers that can introduce single or multiple attachment sites to the surface4, 5. The common goal is always the preservation of the biochemical properties of the biomolecule. The immobilization of enzymes onto nanoparticle-sized materials has been reported to positively affect their activity

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Peptides that specifically bind to a surface are also useful for the functionalization of materials. These have been applied not only to surface functionalization and biosensors assembly14, but also to improve the biocompatibility of materials, and to drive biomineralization15. Using phage display, 20 peptides with affinity for hematite have been reported and a conserved Ser-Pro-Ser motif16 was identified. Analogous sequences were found in proteins naturally binding to metal oxides, and especially OmcA and MtrC that bind to Fe2O316. The peptides were selected for binding under mild conditions, e.g. in PBS, and after repeated washing with PBS enriched with the detergent Tween-2016. Protein engineering allows the modification of proteins at specific sites with desired amino acids. Using two different proteins that were subjected to genetic engineering to introduce a novel affinity for hematite, this study investigated the functionalization of hematite nanoparticles with a blue fluorescent protein, C-phycocyanin17 and the copperdependent enzyme laccase from B. pumilus18 (Figure 1). The fluorescent cyanobacterial light-harvesting protein Cphycocyanin (CPC) is safely used as a colorant in food and its consumption has been shown to have antioxidant, antiinflammatory, and neuroprotective properties for humans19, 20. Laccases are multi-copper oxidases found in fungi and bacteria that catalyze the four electron reduction of O2 to H2O while oxidizing aromatic substrates21. In this study, the thermostable laccase (LACC) from Bacillus pumilus is used18. An enzyme highly similar to LACC has also been reported to have bilirubin oxidase activity22, e.g. with only 6 out of 510 amino acids difference between the one reported by Reiss et al18. Enzymes such as laccases are used in various biocatalytic processes and their immobilization to a solid support can enable their reuse in successive catalytic processes23. Laccases have also been immobilized, in either covalent or not covalent ways, to different substrates such as magnetic silica particles24 and multi-walled carbon nanotubes25, and applied to the development of bio-electronic devices such as sensors for phenolic compounds26-28, medically relevant compounds such as adrenaline29 and morphine30, as a reporter in bioresponsive materials31. Aiming at developing a direct process for the functionalization of hematite with biomolecules, we thus engineered the light-harvesting protein CPC and the oxidoreductase LACC to carry an N-terminal peptide sequence with affinity for hematite and characterized the protein-NP hybrid material.

Figure 1 Schematic view of the process for the affinitybased functionalization of hematite nanoparticles with selected proteins and evaluation of the enzymatic activity of free vs. immobilized protein.

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EXPERIMENTAL PROCEDURES Materials, chemicals and proteins Wild-type C-phycocyanin from Spirulina sp. (Arthrospira sp., CPC, product nr. P2172), Tobacco Etch Virus nuclear inclusion a endopeptidase (TEV protease) (product nr. T4455), and all the chemicals were purchased from Sigma Aldrich (Buchs, Switzerland). Synthesis of hematite nanoparticles Iron oxide nanoparticles (NP) were synthesized using high purity (99%) iron (III) nitrate [Fe(NO3)3.9H2O] and oleic acid [C18H34O2]32. First, oleic acid was heated to 70°C and iron nitrate salt was added to the reaction bath with consequent stirring for 90 min by setting the temperature at 125°C. This step continued until and unless all NO2 fumes is released and a reddish brown viscous mass formed. The as formed viscous mass was dried under liquid nitrogen and dispersed in THF (Tetrahydrofuran). The dispersion was further centrifuged at 5000 rpm for 10 min and the pellet was air dried for 24 hours in a chemical hood. The as dried powder was further subjected to a heat treatment at 500°C for holding time of 30 min in an electrically heated furnace to obtain the hematite phase. The heat treatment was performed in a non-isothermal way in Al2O3 crucibles with Ceram-Aix furnace (FHT 175/30) using a heating rate of 300 K/h. Particle size was determined by FESEM (Hitachi S-4800 model) and surface area by BET method (Coulter SA3100 series surface area and pore size analyzers). The crystallographic properties of hematite were verified by powder XRD analysis (PAN analytical X”Pert PRO, Cu Kα radiation). Cloning of the engineered CPC and LACC variants Plasmid pBS414V17 carried the gene coding for HisCPC17 and was first used as template in a PCR with oligonucleotides (Table S1) to produce the differently tagged versions of CPC. Plasmids pGF023 and pGF024, containing the gene coding for HemCPC and HemHisCPC respectively, were obtained by cloning via isothermal assembly. After digestion with NcoI and EcoRI, the pBS414V vector backbone was isolated. Isothermal assembly reactions were set up by incubating 50100 ng of gel-purified CPC-coding amplicon with the digested vector for 15 min at 50°C33. 1 µl of the isothermal assembly reaction mix was used to transform chemo-competent E. coli DH5α cells. Plated on selective LB agar plates (0.1 mg/ml spectinomycin and 50 µg/ml kanamycin), few clones were selected and the constructs confirmed by sequencing. To ensure the synthesis and covalent attachment of the bilin cofactor, positive constructs were co-transformed with plasmid pAT101 in E. coli DH5α and used for the recombinant production of the CPC variants. Similarly, cloning by isothermal assembly was performed to introduce an N-terminal Hem-tag to LACC from Bacillus pumilus. The plasmid pBul containing the coding sequence for LACC was used as template18 to introduce the N-terminal Hem-tag sequence and TEV cleavage site to LACC by PCR (Table S1). The obtained amplicon was combined with the pBul backbone that was previously digested with NdeI and HindIII in an isothermal assembly reaction. The mix was incubated at 50°C for 15 min and used (1 µl) to transform E. coli DH5α. After confirming the correctness of the construct by sequencing, the


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plasmid was isolated and used to transform the E. coli Rosetta 2 strain that was used for expression.

Protein Database35. Surface potential was analyzed using the plugin APBS, the Adaptive Poisson-Boltzmann Solver36.

Overexpression and purification of CPC variants Glycerol stocks of E. coli DH5α strains carrying plasmids pAT101 and pBS414V, pGF023, or pGF024 were used to inoculate 100 ml LB medium containing 50 µg/ml kanamycin and 80 µg/ml spectinomycin and incubated overnight at 37°C with shaking. Diluted into 2 l flasks containing 500 ml of selective TB medium for an OD600 of 0.04, IPTG was added at a final 1 mM concentration to induce expression, once cultures reached an OD600 above 1. After incubation overnight at 30°C, 180 rpm, cells were harvested by centrifugation, subjected to lysozyme treatment, broken by sonication, and the cell-free extract was separated by ultracentrifugation essentially as in34. Partial purification of HisCPC and HemHisCPC was carried out by immobilized metal ion affinity chromatography (IMAC) with an Äkta Purifier system (GEHealthcare) using 100 mM potassium phosphate pH 7.5, 150 mM NaCl, 5 mM imidazole as buffer. Elution of the protein from the nickelchelated Histrap columns was promoted by increasing imidazole concentrations in a 5-500 mM concentration. Eluted fractions with a visible blue color were pooled, buffer exchanged and stored as aliquots at -20°C. Overexpression and partial purification of HemLACC The expression of the HemLACC variant was carried out as reported in18. Briefly, a preculture of 100 ml culture volume was set up in LB medium supplemented with ampicillin (0.1 mg/ml) and chloramphenicol (30 µg/ml) and incubated overnight at 30°C, 160 rpm. The preculture was diluted in 500 ml of selective LB medium for an initial OD600 = 0.04, incubated at 30°C until an OD600 of 0.4 was reached. Expression was induced by adding 1mM IPTG (final conc.) and 0.25 mM Cu2SO4 (final conc.). The incubation temperature was then lowered to 25°C for 4 h and the flasks incubated at 25°C under static conditions overnight. Cell harvesting and cell-free extract preparation was carried out as previously described for the CPC variants. For partial purification, the cell-free extract containing HemLACC was loaded on a 5 ml His-trap column previously equilibrated with 20 mM Tris pH 7.6 using an Äkta Purifier (GE Healthcare). Elution was carried out with a linear gradient of imidazole from 0 to 1 M over 12 column volumes. Fractions with laccase activity were pooled, concentrated, and the imidazole was removed using a ultrafiltration centrifugal device (10000 cutoff), and stored as aliquots at -20℃. Protein quantification and biochemical characterization Purified and crude preparations of all CPC variants were analyzed by SDS PAGE (16% acrylamide) and UV-Vis spectroscopy in 96-well plate at 22°C. Fluorescence measurements were performed in black 96-well plates (λex=609 nm, λem=400–700 nm). Protein concentration was determined using an extinction coefficient of ε1%620 = 70 for CPC variants and of 77303 M-1cm-1. Protein structure analyses The software Pymol (The PyMOL Molecular Graphics System, Version 1.7.4 Schrödinger, LLC) was used to analyze the three-dimensional structure of LACC, modelled on the available structure of CotA from B. subtilis (PDB ID: 1GSK), and CPC (alpha subunit, PDB ID: 4F0T) as retrieved from the

TEV-catalyzed cleavage of the affinity tags Tag-less CPC versions of each variant were obtained by digestion with TEV protease. A 0.5 mg/ml solution of each protein variant was prepared in PBS and added of DTT (1 mM final concentration) and EDTA (0.05 mM final concentration). TEV-free solutions were also prepared and similarly added of EDTA and DTT. Proteolysis was carried out overnight at room temperature. Cleavage was confirmed by SDS PAGE. The commercial CPC from Spirulina sp. was used as reference. Immobilization of CPC variants to hematite NPs, previously redispersed in PBS, were incubated in the presence of CPC molecules at 22˚C under agitation. For the immobilization study, 10 mg of NPs were incubated in the presence of CPC variants at a concentration of 0.4 mg/ml (Vtot=100 µl). Reactions were prepared in PBS and incubated for 3 h shaking at room temperature (~22°C). NPs were then harvested by centrifugation (5 min at 19357 ×g), the unbound soluble fraction was removed for analyses, and the NPs were rinsed by resuspension in PBS supplemented with 0.5% Tween-20 twice. The zeta potential of the functionalized NPs was determined using a Zetasizer Nano ZS90 (Malvern Instruments) after diluting them in ultrapure water for a final concentration of 0.2 mg/ml. The presence of CPC on the NPs was detected in solution by fluorescence (λex =609 nm, λem =640 nm) in black half-area 96-well plates at room temperature using a 100 µL sample. Immobilization of HemLACC to hematite The incubation of IMAC-purified HemLACC (0.3 mg/ml) with hematite NPs at increasing NP concentration in a range of 0 – 30 mg/ml was carried out in 20 mM Tris buffer pH 7.6 at RT for 3 h in a total volume of 100 µl. An initial stock solution of 200 w/v mg/ml hematite NPs in 20 mM Tris buffer pH 7.6 was used for sample preparation and all reactions were carried out in triplicates. NPs at a 30 mg/ml concentration were used as a negative control and HemLACC without NPs was used in the same conditions as positive control. All the unbound HemLACC was collected from the solution by centrifugation at 22℃ for 10 min at 4000 rpm and the activity was measured. The HemLACC-modified NPs were redispersed in 20 mM Tris buffer pH 7.6 and kept on ice until enzymatic activity measurements. For XPS measurements, the modified NPs were harvested by centrifugation and re-suspended in water twice before being freeze-dried and analyzed. Protein identification Separated by SDS PAGE, a selected band was excised, cut in small pieces, rinsed twice in 100 µl of 100 mM NH4HCO3/acetonitrile (1:1) and once with acetonitrile only. After the addition of 10 µl of trypsin (5 ng/µl) and 30 µl of 10 mM Tris, 2 mM CaCl2 pH 8.2 the sample was incubated for 30 min at 60°C. Peptides were then extracted from the gel using 150 µl of 0.1% TFA/50% acetonitrile for 15 min in an ultrasonic bath. Dried and resuspended in 20 µl of 0.1% TFA, peptides were desalted using ZipTip C18, and analyzed by MALDI/MS/MS. The peptide profile produced was used for identification using the Mascot (uniref100) database. The procedure was performed by the Functional Genomic Center Zurich (Zürich, Switzerland)



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Laccase activity measurement and kinetic study Enzymatic activities were performed using the ABTS oxidation assay as described by Reiss et al.2 Briefly, 10 µl of enzyme-containing solution was incubated with 190 µl of 0.5 mM ABTS in McIlvaine buffer pH 4 (Vtot= 200 µl) and the absorbance at 420 nm was monitored over time at 22℃ using a Cary Bio spectrophotometer (Agilent technologies). In order to determine the kinetic parameters of HemLACC in the free or immobilized form, the activity of the enzyme and of the NP suspension was measured at different concentrations of ABTS (0.1–12 mM, ε=36,000 M-1cm-1) in McIlvaine buffer pH 4 at 22°C. The kinetic parameters were extrapolated using the Michaelis-Menten equation with the Enzyme Kinetics package of Sigma-Plot (Systat Software, Inc., San Jose California USA, www.sigmaplot.com). One unit is defined as the amount of HemLACC required to oxidize 1 µmol of ABTS per minute at 22°C. X-Ray Photoelectron Spectroscopy (XPS) The chemical composition of the surfaces, especially the nitrogen to iron concentration ratio, was analyzed by XPS measurements. They were performed with a Scanning XPS Microprobe (PHI VersaProbe II spectrometer, Physical Electronics) using monochromatic Al Kα radiation (1486.6 eV). The operating pressure of the XPS analysis chamber was below 5 x 10-7 Pa during the measurements. The spectra were collected at photoemission take off angles of 45o (with respect to the sample surface). Survey scan spectra (0 – 1100 eV) were acquired with an energy step width of 0.8 eV, acquisition time of 160 ms per data point and analyzer pass energies of 187.85 eV. Higher resolution narrow spectra for the elements carbon C1s (278 - 298 eV) and nitrogen N1s (391 - 411 eV) were acquired with energy step widths of 0.125 eV, acquisition times of 0.96 s (carbon) and 1.92 s (nitrogen) and analyzer pass energies of 29.35 eV. Under these experimental conditions (pass energies), the energy resolution (FWHM, full width at half maximum height) measured on the silver Ag3d5/2 photoemission line is 2.4 eV and 0.7 eV, respectively. The total acquisition times were approximately 4 min for survey scans and 10 min for the two high-energy resolution elemental scans together, which achieve an adequate signal-to-noise ratio without observable X-ray radiation damage to the samples. Each sample was analyzed at a randomly chosen spot using a micro-focused, scanned X-ray beam with a diameter of 100 µm (operated at a power of 25 W at 15 kV). The 180o spherical capacitor energy analyzer was operated in the fixed analyzer transmission mode (FAT). The samples were pressed onto an indium foil to have a homogeneous and flat sample surface. In order to compensate possible sample charging, dual beam charge neutralization with a flux of low energetic electrons (1.4 eV) combined with very low energy positive Ar-ions (10 eV) was used. The binding energy is referenced to the C-C, C-H hydrocarbon signal C1s at 285.0 eV. The calculation of the atomic concentrations was performed via curve fitting (least-squares fit routines) with CasaXPS software version 2.3.16. Thereby, a mixed Gaussian-Lorentzian product function (constant ratio of 70% Gaussian and 30% Lorentzian) was used to de-convolute the XP spectra and a Shirley type background was subtracted from the XPS peak areas. Tabulated PHI sensitivity factors37 corrected for our system’s transmission function and spectrometer geometry (asymmetry function) have been used for quantification.

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RESULTS AND DISCUSSION Biomolecule engineering In this work, we investigated the use of an affinity peptide specific for hematite for the immobilization of the fluorescent protein CPC and the oxidoreductase LACC. When immobilizing biomolecules to materials, various factors drive the interaction. In general, opposite surface charges and similar hydrophobicity degrees can promote the interaction38. CPC and LACC were first compared on the basis of size, of pI, and of the presence of charged amino acids (Table S2). Under the tested conditions, both CPC and LACC carry a net negative charge. While the surface of CPC presents a mainly positively and a mainly negatively charged region, the charge distribution on the LACC surface shows a minor positively and a major negatively charged region (Figure S1). The α-subunits of C-phycocyanin from Synechocystis PCC 6803 and LACC from B. pumilus were engineered to carry an N-terminal peptide with affinity for hematite, so-called Hemtag (sequence: STVQTISPSNH). The N-terminus of both CPC and LACC is surface exposed, and was thus selected as the site of insertion of the Hem-tag in order to avoid possible interference with the maturation of the protein, e.g. cofactor binding, fluorescent properties, and catalytic activity. Cloning was carried out by isothermal assembly, and the engineered forms of CPC were recombinantly produced in E. coli. Purification by IMAC was carried out for the HisCPC and HemHisCPC, whereas and HemCPC was used as a crude preparation as cell-free extract. Similarly, a Hem-tagged version of the laccase from B. pumilus was engineered and recombinantly produced in E. coli. All constructs were designed in order to allow the removal of the affinity tag by treatment with TEV and the independent evaluation of its impact on protein immobilization (Figure 2).

Figure 2 (A) CPC and (B) LACC variants used in this study differing in the N-terminal sequence. The available structure of CotA (PDB: 1GSK) is used to represent the B. pumilus LACC (68% sequence identity). Cofactors are shown as sphere and the engineered N-terminal peptide is symbolized by a black line.


Effect of the affinity peptide on CPC immobilization All engineered CPC variants showed the sharp absorption peak characteristic of the α-subunit at 620 nm (Figure 3A), as compared to the absorption peak at 615 nm of the wild-type CPC protein that is a result of the presence of the second betasubunit with absorption maximum at 608 nm. In order to assess the impact of the Hem-tag, and possibly His-tag, tagfree versions were obtained from the corresponding CPC variants by TEV cleavage. SDS PAGE analysis showed a lower molecular mass of the TEV-treated proteins (Figure 3B), agreeing with the mass of the removed N-terminal peptide, e.g. calculated mass of 3990.2, 3167.4, and 2895.0 for HemHisCPC, HemCPC, and HiscPC, respectively. Treatment with TEV did not affect the absorptive and fluorescent properties of the proteins (Figure 3, Table S3). 2.5

CPC HisCPC HemCPC HemHisCPC

2

0.12

2.0

80

0.07

CPC-fluorescence of NPs (FL640) Unbound CPC (A620) N/Fe

1.5 0.02

1

500

600

0.5 260

360

460 560 660 Wavelength (nm)

60

700

0

A

fluorescence at 640 nm of the NPs carrying the tagged version of CPC, as compared to the TEV-treated equivalent, was due to a higher presence of protein, as shown by the N/Fe ratio measured by XPS and by SDS PAGE analyses (Figure S2). The increased affinity for the material is thus given by the Hem-tag, as compared to the only adsorption process that can take place in the TEV-treated versions and that depends only on the presence of specific residues on the protein surface. Interestingly, the effect due to His-tag and Hem-tag on the protein binding to the hematite NPs was similar, although due to a different binding mechanism, e.g. based on a conformational basis for the Hem-tag16 and probably electrostatic interaction or hydrogen bonding for the His-tag (Figure 4). The lower binding of CPC carrying Hem- and Histag in tandem to the NPs might be due, for example, to the pairing of the two tags with each other, thus preventing the binding, or to a loss of the conformational freedom that is required for the binding of the Hem-tag due to the additional interaction of the His-tag with the NP surface.

760

FL640

Absorbance

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1.5

40

1.0

20

0.5

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0.0 +TEV -TEV +TEV -TEV +TEV -TEV +TEV -TEV HemCPC HemHisCPC HisCPC

B Figure 3 (A) UV-Vis spectrum of CPC variants in purified (CPC, HisCPC, HemHisCPC) or crude preparation (HemCPC). (B) TEV-catalyzed cleavage of the N-terminal affinity peptides from the CPC variants. SDS PAGE analysis of HisCPC, HemCPC, and HemHisCPC after treatment with TEV stained with zinc acetate (upper gel) and Coomassie (lower gel). Molecular weight markers are reported on the left in kDa. Affinity of the different variants of CPC for hematite was assayed using hematite NPs in a PBS solution at pH 7.2, under the optimal binding conditions16. TEV-treated and tagged HemCPC, HemHisCPC, HisCPC were then incubated with hematite NPs, and fluorescence and UV-Vis spectroscopy were used to assess how the CPC molecules distributed between the solution and the solid surface. After 3 h of incubation, analysis of the unbound fraction showed a higher presence of the untagged TEV-treated forms, in comparison to the tagged HemCPC, HemHisCPC, and HisCPC (Figure 4). NPs incubated with the TEV-treated versions of HemCPC, HemHisCPC, or HisCPC exhibited an 8, 2, and 4-fold lower fluorescence at 640 nm, respectively, than the NP incubated in the presence of the tagged proteins. No effect due to TEV treatment was seen for the untagged commercial version of CPC that was used as control (Figure 4). The increased

CPC

Hem

Figure 4 Effect of affinity tags on the immobilization of HemCPC, HemHisCPC and HisCPC, in comparison to the wild-type CPC to hematite NPs (Hem). Tag-free versions of the proteins were prepared using TEV and subsequently incubated with the NPs. The presence of CPC in the unbound liquid fraction was detected by the absorbance at 620 nm (blue bars), and the NP were analyzed for CPC fluorescence (white bars). NPs were then analyzed by XPS to determine the iron to nitrogen ratios (dots). Additional info in Figure S2 and S3. In order to further study the affinity of the different CPC variants to the hematite NPs, these were incubated in the presence of the tagged or TEV-treated CPC variant in a concentration ranging from 0 to 0.3 mg/ml (15 µM). The treatment with TEV affected the immobilization of all CPC variants and at the highest concentration tested, e.g. 0.3 mg/ml, NPs incubated with the tagged version of HemCPC and HemHisCPC acquired a three-fold higher fluorescence as compared to the TEV-treated equivalents. The advantage of CPC carrying the affinity tag was also visible at low CPC concentrations as the tagged version could be detected on the NPs at 0.3 vs. 3 µg/ml for HemHisCPC, at 3 vs. 300 µg/ml for HemCPC, and at 7 vs. 30 µg/ml for HisCPC in the tagged and TEV-treated versions, respectively (Figure S4). The effect of the Hem-tag could also be quantified as ratio between the fluorescence of NPs incubated with the tagged vs. not tagged version of the CPC variants under identical conditions (Figure


A620 (dil 100X) or N/Fe

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5A). Values of 3.5 for HemHisCPC, 4 for HemCPC, and 2 for HisCPC at the highest CPC concentration tested were obtained. NPs incubated with the crude preparation containing HemCPC interacted also with E. coli intracellular proteins, as peptide mass fingerprinting analyses identified the lactose operon repressor lacI (39 kDa), elongation factor Tu (43 kDa), and the succinyl-CoA synthethase beta chain (41 kDa). Interestingly, the first two proteins are often co-purified with His-tagged proteins during IMAC purification39, 40. The presence of proteins on the NP surface contributed to form a visibly more stable suspension and the zeta potential increased from -50 mV towards less negative values, e.g. ~35 mV for NPs carrying HemCPC (Figure 5). Although the presence of the hem-tag affected the amount of protein immobilized and the overall particles fluorescence, no significant difference in zeta-potential was measured between NPs that were incubated with tagged or TEV-treated protein.

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According to an in silico model of the LACC from B. pumilus, the protein has a largely negative surface potential (Figure S1) that might provide repulsion to the negatively charged hematite NPs surface and thus promote interaction with the surface mainly through the Hem-tag. Due to the previously detected interaction of the His-tagged CPC with hematite, we investigated whether a Hem-tagged protein would bind to a material usually used for IMAC purification, i.e. a Ni2+-chelated resin. A partially purified HemLACC preparation was obtained using a Ni2+-chelated resin commonly used for affinity purification of His-tagged proteins. HemLACC tightly bound the resin and was eluted at an imidazole concentration of 0.6 M. The identity of the protein was confirmed by peptide-map fingerprinting with 23.1% sequence coverage (Figure S5). This interaction was, however, detected also for the untagged version of HemLACC, suggesting a general affinity of the LACC molecule for the resin due to the presence of surface exposed histidine residues (Figure S6). The immobilization of the partially purified HemLACC was first assessed incubating a constant amount of HemLACC in the presence of different concentrations of NPs, i.e. up to 30 mg/ml. We could thus determine that a 5 mg/ml NP concentration equivalent to 1 nmol HemLACC/mg NP was sufficient to achieve a 100% immobilization yield, as no laccase activity was detected in solution. An increase in NP concentration corresponded to an increase of the total laccase activity. Up to a 6-fold increase in total laccase activity was detected in the immobilized fraction at the highest tested NP concentration of 30 mg/ml (Figure 6), e.g. the total initial activity of 2 mU increased to 12 mU (Figure S7).

A

A

B

B Figure 5 (A) fluorescence (λex=609 nm, λem=620-700 nm), and (B) zeta-potential measurement of hematite NPs incubated with HemCPC, HemHisCPC and HisCPC at different concentrations prepared in PBS pH 7.2 (the corresponding SDS PAGE analysis is in Figure S4). In B, the protein concentration is reported as mg/ml on the x-axis and data related to TEV-treated CPC variants are represented as empty dots whereas and the reference untreated NP as a triangle. C

Immobilization of HemLACC and the effect on the catalytic activity The advantages offered by the Hem-tag when CPC immobilization was carried out, were also investigated for a protein with catalytic activity. LACC was selected as model enzyme and engineered to carry an N-terminal Hem-tag.

Figure 6 Effect of immobilization to hematite NPs on the activity of HemLACC. (A) Activity of HemLACC-NPs complexes with different protein/NP ratios. HemLACC (0.3 mg/ml) was incubated in the presence of NPs at different concentration (0-30 mg/ml) and the ABTS-oxidizing activity fraction was measured. The activity measured in the unbound (black line with triangles) and immobilized fraction (red line with dots), is reported. (B) Relative


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ABTS-oxidizing activity of HemLACC in free (red triangle) and immobilized (blue diamonds) form under different pH conditions (experimental values in Figure S8). (C) Schematic illustration suggesting how a decreasing protein/NP ratio would promote the formation of a submono- rather than multi-layer of proteins at the surface, which was associated with an increased enzymatic activity.

The different protein/NP ratio was also measured as N/Fe ratio by XPS with ratios of 0.112 vs. 0.057 for the samples incubated with 20 or 30 mg/ml NP, respectively. No nitrogen was detected for the NP incubated in the absence of protein. With a calculated projection area of 21.47*10-18 m2 for HemLACC, a particle size of ~80 nm (Figure S3) and a surface area of 11 m2/g, for the NPs, HemLACC was estimated to assemble into the equivalent of 3 monolayers (n=3) at the NP surface at the lowest NP concentration (2.55*10-9 mol HemLACC/mg NP) while an enhancement in activity is associated with a n1) is formed. A high protein density at the surface is thus deleterious for the enzymatic activity, possibly because of an altered protein conformation due to steric hindrance and lower degrees of

The project was funded by the VELUX Foundation (project no. 790, BioPEC) and by SERI within the COST action TD1102 ‘Photosynthetic proteins for technological applications: biosensors and biochips (PHOTOTECH)’.

ACKNOWLEDGMENT The scientific support of Artur Braun and Julian Ihssen was appreciated. The technical assistance of Thomas Ramsauer, Luzia Wiesli, and Mahabubur Chowdhury is acknowledged. We are grateful to Prof. Wendy Schluchter for providing the plasmids used for the expression of HisCPC.

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Affinity-Driven Immobilization of Proteins to Hematite Nanoparticles

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