Designed Modular Proteins as Scaffolds To Stabilize Fluorescent

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Designed modular proteins as scaffolds to stabilize fluorescent nanoclusters Pierre Couleaud, Sergio Adan-Bermudez, Antonio Aires, Sara H. Mejias, Begona Sot, Álvaro Somoza, and Aitziber L. Cortajarena Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01147 • Publication Date (Web): 04 Nov 2015 Downloaded from http://pubs.acs.org on November 6, 2015

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Designed modular proteins as scaffolds to stabilize fluorescent nanoclusters Pierre Couleaud,ab‡ Sergio Adan-Bermudez,a‡ Antonio Aires, a Sara H. Mejías,ab Begoña Sot,ab AlvaroSomoza,ab* and Aitziber L. Cortajarena.ab* a

IMDEA-Nanociencia, Campus de Cantoblanco, 28049 Madrid, Spain.

b

Centro Nacional de Biotecnología (CNB-CSIC) - IMDEA Nanociencia Associated Unit,

Campus de Cantoblanco, 28049 Madrid, Spain. *To whom correspondence should be addressed: E-mail: [email protected]; [email protected] ‡

These authors contributed equally to this work.

KEYWORDS: protein design, protein scaffold, repeat proteins, fluorescent nanoclusters, sensors, nanotechnology, Hsp90 binder, bionanotools

ACS Paragon Plus Environment

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ABSTRACT.

Proteins have been used as templates to stabilize fluorescent metal nanoclusters thus obtaining stable fluorescent structures, and their fluorescent properties being modulated by the type of protein employed. Designed consensus tetratricopeptide repeat (CTPR) proteins are suited candidates as templates for the stabilization of metal nanoclusters due to their modular structural and functional properties. Here, we have studied the ability of CTPR proteins to stabilize fluorescent gold nanoclusters giving rise to designed functional hybrid nanostructures. Firstly, we have investigated the influence of the number of CTPR units as well as the presence of cysteine residues in the CTPR protein, on the fluorescent properties of the protein-stabilized gold nanoclusters. Synthetic protocols to retain the protein structure and function have been developed, since the structural and functional integrity of the protein template is critical for further applications. Finally, as a proof-of-concept, a CTPR module with specific binding capabilities has been used to stabilize gold nanoclusters with positive results. Remarkably, the protein-stabilized gold nanocluster obtained combines both the fluorescence properties of the nanoclusters and the functional properties of the protein. The fluorescence changes in nanoclusters fluorescence have been successfully used as sensor to detect when the specific ligand was recognized by the CTPR module.

Introduction


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Gold nanostructures have been deeply studied in the last 20 years mainly due to the striking optical properties of nano-sized gold compared to bulk gold.1, 2 Gold nanoparticles (AuNPs) have a characteristic plasmon absorption band and are mostly developed for sensing applications3 or as nanocarriers in nanomedicine.4-6 Further studies have been focused on the synthesis and stabilization of gold nanoclusters (AuNCs) (diameter < 2-3 nm), which optical properties also differ from AuNPs.7 Particularly, AuNCs have fluorescence properties strictly related to their size due to the confinement of gold atoms.8-11 AuNCs have shown an immense potential in sensing and biolabeling because of their fluorescence properties.12-16 These structures are composed of a few gold atoms and can be stabilized by different molecules such as DNA,17 dendrimers,18 small molecules,19 and proteins.20 In this sense, the protein-directed synthesis and stabilization of gold nanostructures have been studied for years after Brown’s pioneering work in 1997.21 Protein-stabilized AuNCs offer unique properties since the structures obtained are stable under a wide range of pHs and ionic forces, making them ideal for biological applications. Different proteins such as bovine serum albumin (BSA),22 human transferrin,23 ferritin,24 trypsin,25 pepsin,26 horseradish peroxidase,27 insulin,28 and lysozyme29-31 have been employed in the preparation of AuNCs. Nevertheless, most of the published contributions utilize BSA because of its availability and there are not reports involving the use of engineered proteins. The fluorescent properties of protein-stabilized AuNCs are still not fully understood and depend on different factors including the protein employed, the presence of particular amino acids such as cysteines, the local environment of the gold coordination site, and the size of the protein.32 However, up to now protein-based nanocluster synthesis has been done using commercially available globular proteins, which properties are not easily tuned, therefore limiting their applicability in different research areas.


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In this work, we explore the potential of designed repeat proteins as templates for nanocluster synthesis and stabilization (Figure 1). Repeat proteins have become an attractive target for nanotechnology applications due to their modularity.33-37 Repeat proteins contain small structural motifs organized in tandem arrays that lead to extended repetitive structures. Their structural properties make them interesting substrates for the preparation of nanoclusters and further development of more complex functional structures. In particular, we focused on the tetratricopeptide repeat (TPR) module, a 34 amino acids helix-turn-helix motif.38 Consensus TPR proteins (CTPRn) comprise tandem arrays with different number (n) of an identical consensus sequence (Figure 1.).39, 40 These repeat proteins present a modular structure which allows for the engineering of their function41 and stability.42 CTPR proteins have been used as building blocks to generate different nanostructures,37 and ordered materials.36 The CTPR proteins can be produced and purified at large scale following simple molecular biology protocols. Therefore, CTPR units can be conceived as modular units capable of assembling complex functional nanostructures and materials.43 In addition, the potential ability of CTPR units to stabilize nanoclusters will considerably expand their applicability in the field of sensing and imaging. Recently, CTPR protein-mediated synthesis of AuNPs has been reported.44 In the present study, we explore the capability of CTPR proteins to act as templates for the synthesis and stabilization of AuNCs and study to what extent the number of repeats affects the formation of AuNCs by using four CTPR proteins with 3, 6, 8 and 20 repeated modules (named C3, C6, C8 and C20). In addition, we evaluate the effect of a single cysteine residue at the C-terminal end of the different CTPR modules (so-called C3-Cys, C6-Cys, C8-Cys and C20-Cys), as well as a variety of reducing conditions.


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Figure 1. Schematic representation of CTPR repeat modules and the synthetic strategy to prepare CTPR protein-stabilized fluorescent AuNCs. A CTPR unit is represented by orange ribbons and tyrosine residues (six) by sticks. CTPR3 protein structure is depicted in the right panel along with each identical repeat unit colored differently (orange, green, blue, and the Cterminal solvating helix in magenta) (PDB ID: 1NA0). Bottom panel shows a schematic representation of the strategy used to obtain CTPR-AuNCs complexes. CTPR3 represented as ribbons, gold salts as yellow spheres and blue-emitting AuNCs as clusters of blue spheres. Remarkably, the properties of the CTPRs can be easily modulated, such as the binding recognition site hence TPR proteins with the same structure but with different binding activity are created by introducing few variations in the primary sequence.41,

45

Recently, different

Tetratricopeptide Repeat Affinity Proteins (TRAPs) and TRAP-peptide interactions have been designed,46, 47 demonstrating the versatility of the modules. These TRAP modules combined with the fluorescent properties of AuNCs will allow the design of tailored sensors. We investigated the potential of CTPR-stabilized AuNCs as molecular probes by combining the fluorescence of AuNCs and the binding capability of the protein (Figure 1 and 6). For this purpose, it was required to develop a synthetic procedure that allowed the preparation of AuNCs and preserved


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the structural and functional integrity of the CTPR proteins. As a proof-of-concept, a CTPR scaffold that binds the C-terminal peptide of Hsp90 (CTPR390),48-50 a chaperone essential for the folding of many oncogenic proteins and thus involved in tumour progression, was used for the generation of AuNCs. We hereby demonstrated that the CTPR390-AuNCs complex is fluorescent and able to specifically recognize its ligand molecule. Finally, the fluorescence of the coordinated nanoclusters is sensitive to the ligand recognition thus the complex is suitable to be used as a sensor.

Materials and Methods Chemicals All chemicals were purchased from Sigma-Aldrich and used without further purification. Ultrapure reagent grade water (18.2 MΩ, Wasserlab) was used in all experiments. Purification of CTPR proteins CTPR proteins were produced following standard molecular biology protocols for recombinant protein expression. The different CTPRn protein variants with n number of tandem repeats cloned into proEX-HTb vector39, 40, 48 were transformed in Escherichia coli C41 (DE3) cells. The cells were grown in Luria-Bertani media (LB) with ampicillin. The protein expression was induced by IPTG at OD = 0.8. After 5 hours expression at 30°C cells were harvested and the histagged proteins were purified via affinity chromatography using Co-NTA resin. The his-tag was cleaved using TEV protease and a second Co-NTA affinity column purification was performed in order to remove the his-tag and the TEV protease from the protein sample. The protein concentration was determined by absorbance at 280 nm using the extinction coefficient calculated from the amino acid composition. The amino acid sequence of the CTPR3 (C3)


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protein

is

as

follows:

GAMDPGNSAEAWYNLGNAYYKQGDYDEAIEYYQKALELDPNNAEAWYNLGNAYYK QGDYDEAIEYYQKALELDPNNAEAWYNLGNAYYKQGDYDEAIEYYQKALELDPNNAE AKQNLGNAKQKQG. CTPR3-Cys (C3-Cys) presents an additional cysteine residue at the Cterminal (see supporting information). In addition, CTPR390 designed protein was previously described.48 CTPR390 and CTPR3-Cys-90 amino acid sequences are fully detailed in the supporting information. Synthesis of protein-stabilized gold nanoclusters (AuNCs) The protein-stabilized AuNCs were synthesized following a previously reported procedure using NaOH to provide the basic environment needed to reach reducing conditions.22 To investigate the effects of different gold-reducing conditions and the protein sequence, a similar protocol was developed and used for all the hereby described syntheses. 500 µL of 0.3 g/L protein in sodium phosphate buffer (10 mM pH 7.4) were mixed with HAuCl4 (5 µL at 10 mM) for at least 15 min to allow adsorption of gold salts in the protein’s stabilizing sites Then, the reduction of the gold salt to metallic gold was achieved by adding 2 µL 50 mM sodium hydroxide (NaOH) , ascorbic acid, or sodium citrate (2 eq. respect to HAuCl4). The reaction was incubated at 37°C for 18 hours. Finally, the samples were washed several times with water using Amicon ultrafiltration tubes (Amicon Corp., Lexington, MA, USA) with a 10-kDa membrane to eliminate unreacted salts and kept at 4 °C for further experiments. The absorption and fluorescence spectra of protein stabilized gold nanoclusters during the optimization procedure were collected using a Synergy H4 microplate reader (BioTek Instruments, Inc). For the characterization of the C3-Cys-AuNCs complexes the fluorescence and absorption spectra


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(shown in Figure 5) were acquired using a Fluoromax 4 (HORIBA Jobin Yvon Inc., NY, USA) and a Cary 50 Conc (Varian INc, Palo Alto, CA, USA) in quartz cuvettes, respectively. Mass spectroscopy Mass spectra were acquired on an Applied Biosystems Voyager Elite MALDI-TOF mass spectrometer with delayed extraction (Applied Biosystems, Framingham, MA, USA) equipped with a pulsed N2 laser (λ = 337 nm). Sinapic acid was used as matrix. An extraction voltage of 20 kV was used. All mass spectra were acquired in the positive reflectron mode using delayed extraction. Each sample mass spectrum consisted of an average of 50–100 laser shots. MALDITOF sample preparation included 1µL sample suspension mixed with 3 µL of Sinapic acid in 50:50 water/acetonitrile with 0.1% TFA. Then, 1 µL of the mixture was deposited onto the MALDI plate and allowed to air dry. The instrument was externally calibrated using monoisotopic peaks from the sinapic acid matrix (MH+ at m/z 225.071). Transmission electron microscopy (TEM) LaceyCarbon Films on 400 MeshCopperGrids (Agar Scientific) were exposed to a glow discharge before sample deposition. TEM samples were prepared by depositing 10 µL of the sample solution on the grid. After 3 min, the excess solution was removed from the grid using filter paper. To remove the deposited salt, the grid was then washed with a drop of water and the excess water was dried using filter paper. Micrographs were recorded using a JEOL JEM 2100F electron microscope operating at 200KV with a Field EmissionGun equipped with an INCA xsight energy dispersive x-ray radiation (EDX) detector (Oxford Instruments) to analyze the elemental composition of the material. The particle size distribution was determined from TEM micrographs using Image J software measuring the diameter of a total of 72 nanoclusters. Fluorescence quantum yield


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The fluorescence quantum yield (Φx) was calculated using anthracene in ethanol as reference (ΦRef = 0.27, λexc = 370 nm and λem = 423 nm) and the following formula:

where Gradx and GradRef are the gradient from the plot of integrated fluorescence intensity vs. absorbance at excitation wavelength, for the sample and the reference, respectively, and ηx and ηRef the refractive indexes of the solvents, water and ethanol, respectively. Fluorescence measurements were done using a Fluoromax 4 (HORIBA Jobin Yvon Inc., NY, USA) and absorbance was recorded with a Cary 50 Conc (Varian INc, Palo Alto, CA, USA) in quartz cuvettes. Secondary structure CTPR-AuNCs by circular dichroism Upon the generation of gold nanoclusters, the protein secondary structure was examined by circular dichroism (CD) using a Jasco J-815 spectrometer (JASCO Corporation, Tokyo, Japan). CD spectra were acquired at 10 µM protein concentration in a 0.1 cm path length cuvette using a 1 nm band-width with 1 nm increments and 10 seconds average time. Binding and sensing properties of CTPR-AuNCs The ligand binding capability of the CTPR protein scaffold upon the generation of gold nanoclusters was studied using the CTPR3-Cys-90 (C3-Cys-90) protein and its target peptide. C3-Cys-90 was designed to bind the C-terminal sequence of Hsp90 protein. 48, 50 The C3-Cys-90AuNCs binding affinity to Hsp90 peptide was determined by fluorescence anisotropy titrating with increasing amounts of C390-AuNCs into a 50 nM fluorescein-labelled Hsp90 peptide solution.51, 52 Fluorescence intensities were recorded in a Fluorolog–TCSPC spectrofluorometer


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(Horiba) equipped with excitation and emission polarizers. Excitation was accomplished with a 5 nm slit-width at 492 nm and the emission recorded at 516 nm with slit widths of 5 nm. The sensing properties of C3-Cys-90-AuNCs were studied monitoring the AuNCs fluorescence. The fluorescence emission spectrum of a 20 µM protein concentration in PBS of a C3-Cys-90-AuNCs solution was recorded. The change on the fluorescence emission spectra was determined upon titration with Hsp90 ligand peptide (from 5 to 400 µM). As control we used a non-cognate peptide that is not recognized by C3-Cys-90-AuNCs. Fluorescence spectra were recorded in a FluoroMax-4 spectrofluorometer (Horiba). Excitation was accomplished with a 5 nm slit-width at 370 nm and the emission recorded from 390 nm to 550 nm with slit widths of 5 nm. The fluorescence intensity was recorded at the maximum of emission (440 nm) and the average of 3 measurements for each point was reported. The error bars represent the standard deviations. The limit of detection (LOD) and limit of quantification (LOQ) of the proof-of-concept sensor were determined based on the standard deviation of the response (SD) of the curve and the slope of the calibration curve (b) according to the following equations: LOD = 3.3 (SD/b) and LOQ = 10 (SD/b).53

Results and Discussion CTPR-stabilized AuNCs First, we tested the ability of designed CTPR proteins to stabilize AuNCs, following a previous protocol described for BSA using sodium hydroxide.22 We studied the influence of the number of CTPR repeats and the presence of cysteine residues in the CTPR protein, on the fluorescent properties of the protein-stabilized gold nanoclusters. As shown in Figure 2, all the CTPR proteins tested were able to stabilize fluorescent AuNCs.


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Figure 2. Fluorescence spectra of AuNCs formed in basic conditions (NaOH) and stabilized by (a) C3, C6, C8 and C20 and (b) C3-Cys, C6-Cys, C8-Cys and C20-Cys. λexc = 370 nm. We observed that the fluorescent properties of the protein-stabilized AuNCs varied depending on the protein length and sequence (Figure 2).54 Figure 2a shows that all the AuNCs obtained using CTPR proteins with 3, 6, 8 and 20 CTPR repeats (C3, C6, C8, and C20) as templates, had the same spectra profile with a strong emission band in the blue region at 420 nm. This result suggests that the CTPR protein size is not critical to stabilize gold nanoclusters. The AuNCs obtained using CTPR proteins containing one Cys residue as templates (C3-Cys, C6-Cys, C8-Cys and C9-Cys) showed different emission spectra profile (Figure 2b).


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Interestingly, C3-Cys showed an emission spectrum with two emission bands at 420 nm (blueemission) and 710 nm (red-emission). By contrast, AuNCs stabilized by C3 only showed blue emission (Figure 2a), highlighting the role of the Cys residue in the generation of red-emitters. Cysteine residues play a fundamental role in the stabilization of AuNCs, as it has been extensively described in the case of BSA.54,

55

Red-emitting BSA AuNCs were described to

comprise around 25 atoms (Au25) and stabilized by the 35 cysteine residues of a single BSA monomer.22 Additionally, it has been reported that at least 5 cysteine residues are required to obtain red and near infrared (NIR) emitters.32 Therefore the stabilization of such Au25NCs by C3-Cys protein might be due to the formation of a surrounding corona of C3-Cys molecules around Au25NCs, as it has been described for glutathione56-58 or other thiol-derivatives.59 Noticeably, the red emission observed in the case of C3-Cys was not obtained for C6-Cys, C8Cys and C20-Cys (Figure 2b). The emission spectra of the AuNCs stabilized by long CTPRs with and without cysteine residues did not show significant differences (Figure 2a and 2b). The absence of red emission can be explained based on the fact that the longer the CTPRs are, the lower the amount of cysteine residues per total number of amino acids is. The reactions were compared at the same mass of total protein and consequently the concentration of cysteines in the reaction was lower for longer CTPRs. Additionally, a higher steric impediment for longer CTPRs might hamper the potential stabilization of AuNCs by several CTPRs through Cys-gold interactions. Nevertheless, the presence of AuNCs emitting in the blue region was observed. Blue-emitting AuNCs have been described to correspond to smaller clusters with less than 20 atoms of gold.26, 60, 61 In this case, the stabilization of metallic clusters might occur because of the tyrosine residues in the protein chain.62, 63 Indeed, CTPR proteins have six tyrosine residues per repeat44 that might stabilize very small AuNCs or else, several molecules could form a


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stabilizing corona around AuNCs.64 The formation of di-tyrosine dimers emitting in the same range could interfere with our results. However, this effect can only take place under highly oxidative conditions where the dimers obtained can be excited at 300 nm with a blue emission at 400 nm,65,

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which is not the case in our experiments with AuNCs. Moreover, control

experiments using C3-Cys with sodium hydroxide in absence of gold salts did not yield any fluorescent material, thus discarding the generation of di-tyrosine dimers as a source of blue fluorescence (Supporting information, Figure S1). Based on these first results, C3-Cys was selected to study the effect of the reducing conditions on the synthesis and stabilization of protein-AuNCs. Three different reducing conditions were tested (NaOH, ascorbic acid 67-69 and sodium citrate70, 71) (Figure 3).

Figure 3. Fluorescence emission spectra of C3-Cys-stabilized AuNCs under different reaction conditions using sodium hydroxide (black line), sodium citrate (red line) or ascorbic acid (green line). λexc = 370 nm. Sodium citrate yielded poor results regarding the generation of AuNCs, showing a lower efficiency than NaOH and red fluorescence emission was not observed (Figure 3). In the case of


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ascorbic acid, AuNCs were obtained with higher yields than for citrate leading to higher fluorescence intensity at 450 nm, comparable to the results obtained when NaOH was used. Under this synthesis conditions red fluorescence was not observed (Figure 3). The fluorescence excitation spectrum showed a maximum at 370 nm and the emission spectra a maximum at 450 nm when excited at 370 nm (Figure 3). In order to ensure that the fluorescence emitters were C3Cys-stabilized AuNCs a complete set of control experiments were carried out (see supporting information, Figure S2). It is clear that the presence of the C3-Cys protein is essential for the stabilization of AuNCs and also that the ascorbic acid is needed for the reduction of gold salts given that fluorescence emission was observed only when all the reagents were present (Figure S2). Moreover, in the case of the control of gold salts mixed only with ascorbic acid, gold nanoparticles were formed (plasmon band at 530 nm observed in visible absorption spectroscopy, data not shown), which gave us an additional evidence of the role of C3-Cys stabilizing AuNCs at first and blocking the formation of large nanostructures later on.

Characterization of CTPR-stabilized AuNCs Due to the interesting results obtained with C3-Cys (Figure 2 and 3) we proceeded to characterize this system in detail. Since we are interested in the structural properties of CTPR repeats, we have tested if the protein secondary structure was maintained after the CTPR-AuNCs synthesis. The CD analysis of the sample treated with NaOH revealed that the protein loses the characteristic CD signal for alpha helical structure (Figure 4). Although the formation of AuNCs was efficiently achieved using NaOH, it is critical to keep the structure of the protein to explore future applications. Noticeably, after the CTPR-AuNCs formation using mild reducing agents (ascorbic acid and


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sodium citrate) the protein structure is retained as it is shown in the circular dichroism spectra (Figure 4). Thus, the optimized AuNCs synthesis protocol using ascorbic acid resulted in C3Cys-AuNCs with good fluorescent properties and the structural integrity of the protein scaffold. We therefore selected this synthesis protocol and C3-Cys as an ideal scaffold for the synthesis and stabilization of AuNCs.

Figure 4. Circular dichroism spectra of C3-Cys-AuNC complexes generated in different reducing media (NaOH, ascorbic acid and sodium citrate). The spectra were acquired in the region that reports the protein secondary structure elements, in particular the alpha-helical structure of the CTPR proteins. The UV−visible spectrum (Fig. 5a) of the protein-stabilized AuNCs (C3-Cys-AuNCs) compared with the spectrum of the protein (C3-Cys) at the same concentration, showed a broad absorption band around 370 nm in addition to the characteristic 280 nm protein absorption peak. The excitation spectrum showed a single peak with a maximum at 370 nm (Fig. 5b). The fluorescence emission spectrum of the product consisted of a single peak with a maximum at 450 nm, when excited at 370 nm (Fig. 5b). In addition, blue fluorescence emission was observed when C3-Cys-AuNCs solution was imaged under UV-light (Figure 5b inset).


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Figure 5. (a) UV-visible spectra of C3-Cys protein (black line) and C3-Cys-AuNCs (red line). The image shows a picture of the C3-Cys-AuNCs solution under visible illumination. (b) Fluorescence excitation (black line) and emission (red line) spectra of C3-Cys-stabilized AuNCs reduced by ascorbic acid. The image shows a picture of the C3-Cys-AuNCs solution under UVlight (360 nm). The fluorescence quantum yield (Φx) of the AuNCs obtained with C3-Cys and using ascorbic acid as reducing agent was 3.5±1.5% in water. This Φx is lower than Φx of previously reported blue emitting PAMAM-stabilized AuNCs in methanol (52%),

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but it is in the same range as

tyrosine-stabilized AuNCs (2.5%), or by pepsin-stabilized AuNCs (3.7%) in aqueous solution. 26, 72


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C3-Cys-AuNCs formation was confirmed using MALDI mass spectrometry. Mass spectra showed two main peaks corresponding to C3-Cys protein (m/z 14496) and a higher molecular weight peak (m/z 17390) that corresponds to C3-Cys-AuNCs complexes with 14 to 15 gold atoms/ per protein (Figure S3). This result is consistent with the blue-fluorescent properties of C3-Cys-AuNCs that have been previously reported for AuNCs with less than 20 atoms73 or in the

presence

of

a

few

smaller

AuNCs

per

protein.

Figure 6. TEM image of AuNCs formed by ascorbic acid and stabilized by C3-Cys.

Finally, TEM images of the C3-Cys-AuNCs showed the absence of gold nanoparticles (d>4 nm) and the presence of only smaller nanostructures of an average of 1.6 ± 0.3 nm (Figure 6). Elemental analysis determined by EDX confirmed the presence of gold nanoclusters and indicated an approximate Au weight content of 5.13 %. The signal due to smaller clusters was very weak and close to the detection limit of the instrument. The absence of gold nanoparticles


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was confirmed by UV-visible spectrum and the characteristic around 530 nm plasmon band was not observed (Figure 5a).

CTPR-AuNCs functional complexes Upon demonstrating the capability of CTPR-AuNCs to maintain the original protein structure, we expect CTPR modules to retain their function. As a proof-of-concept for the design of functional CTPR-AuNCs, a designed CTPR protein encoding a specific binding activity was used as template for the synthesis and stabilization of AuNCs (Figure 7a). In particular, we synthesized CTPR3-Cys-90 (C3-Cys-90), protein that recognizes the C-terminal sequence of the Hsp90 chaperone.48, 49, 74 We obtained fluorescent C3-Cys-90-AuNCs comparable to the clusters obtained for C3-Cys using ascorbic acid as mild reducing agent (see supporting information, Figure S4). First, we used fluorescence anisotropy to monitor the interaction between the C3Cys-90-AuNCs complex and a fluorescein-labeled Hsp90 peptide to determine the corresponding Kd value (Figure 7b). Our results showed that the capability of the protein to recognize the Hsp90 peptide was not affected by the nanocluster formation. The Kd value obtained for the interaction was about 200 µM, comparable to the value obtained using the same assay for the C3-Cys-90 protein.48 This data confirmed that formation of gold nanoclusters did not alter the binding domain, which is key to use this new methodology to develop tools for bioimaging and sensing.


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Figure 7. (a) Schematic representation of CTPR-based sensor. C3-Cys-90 protein is used as template to stabilize fluorescent AuNCs. Upon the recognizing the Hsp90 peptide, the AuNCs local environment, changes leading to an increase in the AuNCs fluorescence emission intensity. (b) Ligand binding activity of C3-Cys-90-AuNCs complex. Fluorescence anisotropy of fluorescein labeled 24-mer C-terminal peptide of Hsp90. The percentage of peptide bound is plotted vs. the protein concentration. The data were fit to a 1:1 binding model to calculate the dissociation constant (KD = 200 µM) (c) Sensing properties of C3-Cys-90-AuNCs complex.


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Change on the fluorescence emission intensity of the AuNCs from C3-Cys-90-AuNCs complex upon the addition of increasing concentrations of Hsp90 peptide (black circles) and in the presence of a non-cognate control peptide (black squares). The emission intensity is recorded at 450 nm (excitating at 370 nm). Each point is the average of 3 measurements and the error bars correspond to the standard deviation calculated for each point. The inset shows representative fluorescence emission spectra at peptide concentrations of 0, 50, 150, and 300 µM. As a first proof-of-concept application, we used the fluorescence of the AuNCs in the C3-Cys90-AuNCs complex as sensor to detect the presence of Hsp90 peptide (non-labelled) (Figure 7a). We observed an increase of the AuNCs fluorescence when Hsp90 peptide was added to a solution containing C3-Cys-90-AuNCs (Figure 7c). The ligand peptide induced a dosedependent increase in the fluorescence of the AuNCs due to the interaction with its cognate peptide (Hsp90) (Figure 7c). A linear response of the fluorescence intensity change was observed in the concentration range of 5-150 µM (Figure 7c and S5), which was fit to determine the limit of detection (LOD) and the limit of quantification (LOQ) of the proof-of-concept sensor. LOD was determined to be 20 µM (46 µg of Hsp90/mL) and LOQ 60 µM (138 µg of Hsp90/mL). When a non-cognate peptide was used in the assay, no significant changes in the fluorescence intensity of the AuNCs were observed, since the CTPR does not recognize it. These data confirm the potential of this approach to build sensors and imaging modules based on CTPR proteins. Noteworthy, the ease of generation of fluorescent AuNCs makes this methodology ideal to develop sensors in straightforward and cost-effective manner if compared to standard labeling techniques such as those that imply the use of organic dyes or chimeras with fluorescent proteins.


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Conclusions This work demonstrates, for the first time, that designed repeat protein scaffolds can be used to synthetize and stabilize gold nanoclusters. It has been shown that neither the number of repeats in the protein, nor the presence of a single cysteine residue interfered with the formation of small blue-emitting AuNCs. A possible explanation is the presence of tyrosine residues in the repeat sequence that could adsorb gold ions and therefore stabilize the gold nanoclusters. The synthesis method using NaOH to generate a reducing environment, proved to be the most efficient. However, highly basic conditions denature the protein and thus destroy its structure and function. This fact can hamper its use in further applications in which structural and functional properties of the proteins are exploited. Therefore, we developed a new synthesis protocol using milder reducing agents such as ascorbic acid and sodium citrate, increasing the efficiency to obtain the AuNCs when ascorbic acid was employed. Whenever reactions did not include the protein scaffold the AuNCs generation did not occur, highlighting the key role of the CTPR proteins as stabilizing agents. The optimized conditions for the AuNCs formation include a metal:protein ratio of 5:1 compared to the conditions previously reported for the CTPR stabilization of AuNPs in which larger ratios were used (1000:1 to 20:1) and smaller the ratios yielded smaller nanoparticles,44 which comes to an agreement with this report where nanoclusters are obtained with smaller metal:protein ratios. Most importantly, this method was mild enough to preserve the structure of the protein and thus permitted to take advantage of the specific function of the protein scaffold. Indeed, we demonstrate that neither the synthesis of the nanoclusters, nor the nanoclusters themselves interfered with the protein binding activity. Thus, we obtained a novel bi-functional designed unit that combines the function of the designed CTPR protein scaffold on one hand and the fluorescence emission of AuNCs on the other. Finally, we demonstrate that the


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fluorescence signal of the protein-stabilized AuNCs is sensitive to the ligand recognition of the specific binding module. This result confirms the potential of this system to develop new sensors. The use of repeat protein scaffolds as biomolecular templates in which stability, size, and function can be tuned at will,42, 45, 75 that also comprise rational methods for the synthesis of fluorescent nanoclusters preserving the protein functions, leaves a door open for further applications in the field of molecular imaging and sensing.

ASSOCIATED CONTENT Supporting Information. Sequence information of the CTPR proteins; controls of the synthesis of CTPR-stabilized AuNCs; mass spectrometry analysis of the CTPR-AuNCs complexes; characterization of the C3-Cys-90-AuNCs complexes; additional information about the characterization of the CTPR-AuNCs sensor. This material is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGMENTS This work was partially supported by the Spanish Ministry of Economy and Competitiveness (BIO2012-34835, SAF-2010-15440, and SAF2014-56763) and the European Commission International Reintegration Grant FP7-PEOPLE-IRG-246688 BIONANOTOOLS (ALC), and IMDEA Nanociencia. SA thanks IMDEA-Nanociencia for financial support through an “Ayuda de Iniciación a la Investigación” fellowship. SHM thanks the Basque Government for financial support (PhD Scholarship). We thank Christian Duarte for his careful editing, corrections and comments on the manuscript.


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ABBREVIATIONS CTPR, Consensus TetratricoPeptide Repeat; AuNCs, gold nanoclusters; Cn, Consensus TetratricoPeptide repeat protein with n number of repeats; Cn-Cys, Consensus TetratricoPeptide Repeat protein with n number of repeats and a single cysteine residue at the C-terminal end; CTPR390 (C3-90), Consensus TetratricoPeptide Repeat protein with 3 repeats designed to bind the C-terminal peptide of Hsp90; C3-Cys-90, CTPR390 protein with a single cysteine residue at the C-terminal end; TRAP, Tetratircopeptide Repeat Affinity Proteins; BSA, bovine serum albumin; CD, circular dichroism; TEM, transmission electron microscopy; LOD, limit of detection; LOQ, limit of quantification.

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For Table of Contents Use Only Designed modular proteins as scaffolds to stabilize fluorescent nanoclusters Pierre Couleaud, Sergio Adan-Bermudez, Antonio Aires,

Sara H. Mejías, Begoña Sot,

AlvaroSomoza, and Aitziber L. Cortajarena. Table of Contents Graphic and Synopsis Designed modular proteins as scaffolds to stabilize fluorescence nanoclusters for the generation of sensing tools.


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Designed Modular Proteins as Scaffolds To Stabilize Fluorescent

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