Chemical Physics Letters 501 (2010) 108–112

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Chemical Physics Letters

journal homepage: www.elsevier .com/locate /cplet t

Size-selective recognition of gold nanoparticles by a molecular chaperone

Masafumi Sakono a,b,⇑, Tamotsu Zako a, Srdja Drakulic c, José María Valpuesta c, Masafumi Yohda d,Mizuo Maeda a

a Bioengineering Laboratory, RIKEN Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japanb PRESTO, Japan Science and Technology Agency, 3-5 Sanbancho, Chiyodaku, Tokyo 102-0075, Japanc Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus de la Universidad Autónoma de Madrid, Darwin, 3, 28049 Madrid, Spaind Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan

a r t i c l e i n f o

Article history:Received 16 August 2010In final form 17 October 2010Available online 21 October 2010

0009-2614/$ - see front matter � 2010 Elsevier B.V. Adoi:10.1016/j.cplett.2010.10.035

⇑ Corresponding author at: Bioengineering LaboHirosawa, Wako, Saitama 351-0198, Japan. Fax: +81 4

E-mail address: msakono@riken.jp (M. Sakono).

a b s t r a c t

Molecular chaperone prefoldin (PFD) assists protein folding by inhibition of protein aggregation in thecell. In this study, we investigated an interaction between gold nanoparticles (Au NPs) and the molecularchaperone PFD. We demonstrated that addition of PFD inhibits salt-induced aggregation of Au NPs. Inter-estingly, the interaction between PFD and Au NPs was observed to be Au-size dependent. As PFD has nothiol groups in the molecule, hydrophobic interaction is considered to be the primary interaction. This isthe first demonstration of size-specific stabilization of Au NPs with non-covalent bonding.

� 2010 Elsevier B.V. All rights reserved.

1. Introduction

There is great interest in the rational self-assembled fabricationof hybrid materials from inorganic nanoparticles and biomolecules[1]. Due to their biocompatibility and tailorability, gold nanoparti-cle (Au NP) conjugates have shown promise as therapeutics [2–4],intracellular gene regulation agents [5–7], and in vitro diagnosticprobes [8]. In recent years, the preparation of Au NP complexeswith proteins has attracted much attention [9]. By combining theintrinsic functionality of many proteins or peptides with Au NPs,the production of nanomaterials with novel function is expected[10]. In an effort to overcome the challenges of preparing pro-tein–Au NP complexes, proteins have been subjected to modifica-tion by adding ligands that can bind metals [11–14]. However,these type of modifications might affect their conformation andfunction. Therefore, proteins with the ability to form complexeswith Au NPs without any ligand modification, are required. Herein,we report for the first time that prefoldin (PFD) [15–20], a molec-ular chaperone with no thiol groups or metal-binding ligand, hasthe ability to interact with Au NPs. It is known that molecularchaperones assist protein folding by inhibiting protein aggregationin the cell. PFD (86 kDa) possess a jellyfish-like structure consistingof six long and protruding coiled-coil tentacles that can interactwith non-native proteins through hydrophobic interactions [21–23]. We demonstrate that addition of PFD inhibits salt-inducedaggregation of Au NPs. More importantly, the interaction betweenPFD and Au NPs is Au-size dependent. Our results suggest that themolecular chaperone PFD can be used as a host protein for Au NPs.

ll rights reserved.

ratory, RIKEN Institute, 2-18 462 4658.

2. Experimental

2.1. Reagents and proteins

Au NPs of various sizes were purchased from British BiocellInternational (UK). Lysozyme and bovine serum albumin were ob-tained from Sigma Chemical Company (USA). PBS solution(137 mM NaCl, 8.1 mM Na2HPO4, 2.68 mM KCl, 1.47 mM KH2PO4)was purchased from Nippon Gene (Japan). All other chemicalswere obtained from the Wako Pure Chemical Company (Japan).P. horikoshii prefoldin was expressed in Escherichia coli BL21(DE3) and purified as previously described [18] (See Figure S1).

2.2. Preparation of the protein–Au NP complex

The purchased colloidal solution containing Au NPs was centri-fuged at 15 000 rpm at 4 �C, and then Au NPs were precipitated in atest tube. The Au NPs smaller than 10 nm and larger than 15 nmwere pulled down by centrifugation for 60 and 20 min, respec-tively. After removing the supernatant from the test tube, a PBSbuffer solution containing protein at various concentrations wasadded into the test tube, and then the Au NPs were dispersed bypipetting immediately. The solution including Au NPs was incu-bated for 10 min at room temperature, and the colloidal sampleswere analyzed by the method described below.

2.3. Measurement of Au NP absorbance by UV–Vis spectroscopy andcalculation of the Au NP aggregation ratio

The absorbance of Au NPs in the range of 400–800 nm was mea-sured using a Cary-50 UV–Vis spectrophotometer (Varian). The AuNPs dispersed in pure water and in the PBS solution enhancing Au

wavelength [nm]400 600 800

Abs.

1a b

c

0LYS BSA PFD

LYS

BSA

PFD

PFD BSALYS

In water

In water

Water PBS

In PBS

Figure 1. (a) Absorption spectra of Au NPs in water or PBS buffer in the presence oflysozyme (LYS), bovine seroalbumin (BSA) and prefoldin (PFD). (b) Photographs ofthe colorimetric change taken place in the Au NPs upon incubation with the sameproteins. (c) TEM observations of the morphology of Au NPs in PBS buffer with thesame proteins. Bar indicates 50 nm.

M. Sakono et al. / Chemical Physics Letters 501 (2010) 108–112 109

NP aggregation were measured at 520 and 620 nm, respectively.Therefore, an absorbance ratio between each wavelength indicatesthe degree of Au NP aggregation.

2.4. Measurement of equilibrium constants of proteins and Au NPs

Various concentration of proteins were mixed with fixed con-centration of Au NPs (10 nM) in PBS and incubated at 10 min.The absorbance was measured by UV–Vis spectroscopy and aggre-gation inhibition ratio was calculated. This ratio reflects theamount of protein–Au NP complex. Kd were determined by fittingto the data using Eq. (1).

½Protein—Au NP� ¼ ð½Protein�½Au NP�Þ=ðKd þ ½Protein�Þ ð1Þ

2.5. Transmission electron microscopy

In the case of unstained samples, aliquots were placed onto acarbon-coated copper grid and allowed to adsorb. Excess samplewas removed from the grid using filter paper, and the grid was leftto dry. Samples were observed with an excitation voltage of 100 kVusing a JEM-1011 transmission electron microscope (JEOL, Tokyo,Japan). In the case of stained samples, aliquots of Au NP either inthe absence or presence of the chaperone PFD, were diluted withMQ water and PBS buffer, respectively, and stained with 2% uranylacetate. Micrographs were recorded at 60 000 magnification in aJEOL JEM1200EXII microscope operated at 100 kV.

2.6. Dynamic light scattering measurement

The DLS experiments were performed using the Zetasizer Nanoinstrument (Malvern Instruments, Worcestershire, UK). About50 ll of solution containing Au NPs was transferred to a 1 � 1 cmfluorescence cuvette (Hellma, Müllheim, Germany), and measuredusing a 4 mW He–Ne laser (633 nm wavelength) with a fixeddetector angle of 173�, under temperature-controlled conditions(at 25 �C with a tolerance of ±0.02 �C).

3. Results and discussions

Firstly, we examined the behavior of Au NPs in buffer solutionwith various proteins including PFD. Dispersed Au NPs have an in-tense surface plasmon band at approximately 520 nm, resulting ina red-colored solution. Upon aggregation of Au NPs by addition ofbuffer, red-shifted extended plasmon bands typically appear at�620 nm, causing a characteristic transition in solution color fromred to purple-violet. This aggregation is induced by the increase ofsurface hydrophobicity with salting-out. Absorption spectra of AuNPs (10-nm diameter) in PBS buffer in the presence of differentproteins (Lysozyme (LYS), Bovine Serum Albumin (BSA) and PFD)were measured by UV–Vis spectroscopy (Figure 1a). In the solutioncontaining LYS or BSA, the absorption maximum wavelengths ofAu NPs were red-shifted by about 30 or 40 nm, respectively, withtheir spectra showing a decrease in the absorption at 520 nmand an increase in absorption at 620 nm. On the other hand, onlya small long-wavelength shift (about 10 nm) with few changes inabsorption at 520 nm and 620 nm was observed for the Au NPsolution with PFD. These spectrum changes of the Au NP solutionupon addition of these proteins were associated with changes incolor (Figure 1b). While the color of the Au NP solution with LYSor BSA changed to purple-violet, a red color which is characteristicof dispersing Au NPs in water was observed for the solution withPFD. These results suggest that PFD has a higher ability to inhibitsalt-induced aggregation of Au NPs than LYS or BSA. Inhibition of

Au NP aggregation was also confirmed by transmission electronmicroscopy (TEM) (Figure 1c). In the solution with LYS or BSA,Au NP aggregates of many sizes were observed. In contrast, noaggregates but dispersed Au NPs were observed in the solutionwith PFD.

Figure 2 shows the influence of protein concentrations on theaggregation inhibition effect of 10-nm Au NPs (see also FigureS2). No inhibition higher than 60% was achieved in the presenceof large LYS concentrations, and over 1 lM BSA was necessary fora high inhibition effect (over 80%), which is consistent with a pre-vious study showing formation of a stable Au NP–BSA (15 lM)complex [24]. In contrast, 100 nM of PFD was enough to preventAu NP aggregation, which suggests a higher affinity between AuNPs and PFD (see also Figure S3). Dissociation constants with 10-nm Au NPs were calculated to be 340 nM for BSA and 30 nM forPFD. Since Au NPs produced by the citrate reduced method havea negatively charged surface and LYS is positively charged in PBSbuffer, it is plausible that LYS could work as salt bridge chemicalbetween Au NPs. This idea was further supported by TEM observa-tions showing large Au NP aggregates in the presence of LYS (Fig-ure 1c). In order to investigate the inhibition mechanism of PFD onAu NP aggregation, the inhibition effect on various diameter sizesof Au NPs (5–20 nm) was examined (Figure 3; the spectra for eachsize are shown in Figure S4). The absorbance at 520 nm for Au NPsover 15 nm in size decreased, while an increase in absorbance at620 nm was not detected as in Figure S4. Furthermore, the produc-tion of Au NP precipitates was confirmed by visual inspection.These results imply that the stability of Au NPs larger than

1 3 50

60

100

Aggr

egat

ion

Inhi

bitio

n R

atio

[%]

Protein Concentration [uM]

40

20

80

Figure 2. The influence of protein concentrations on the aggregation inhibitioneffect of 10-nm Au NPs. Square, triangle and circle symbols represent addition ofPFD, BSA and lysozyme, respectively.

a

b

0

100

50

Aggr

egat

ion

Inhi

bitio

n R

atio

[%]

531PFD Concentration [uM]

Diameter=5 nm

20 nm15 nm

Diameter=10 nm

5 nm

15 nm

20 nm

10 nm

Figure 3. (a) Aggregation ratio with different-sized Au NPs in the presence of PFD atvarious concentrations. (b) TEM observations of Au NP morphology of various sizesin the presence of PFD. Bar indicates 100 nm.

110 M. Sakono et al. / Chemical Physics Letters 501 (2010) 108–112

15 nm in size could not be assessed by the above absorption ratio.Therefore, the degree of decreasing absorbance at 520 nm com-pared with Au NPs in pure water was regarded as the aggregationratio. Figure 3a shows the aggregation ratio at each size of Au NPsin the presence of PFD at various concentrations (see Figure S4). Asshown in the figure, Au NPs with diameter sizes of 5 and 10 nmwere stabilized by PFD in a dose-dependent manner. The dissocia-tion constant between PFD and Au NPs was calculated to be 55 nMfor 5-nm Au NPs and 30 nM for 10-nm Au NPs. On the other hand,the inhibition effect on Au NP aggregation was much weaker for15- or 20-nm Au NPs. This result is also confirmed by TEM obser-vations (Figure 3b) which revealed a clear dispersion of the 5 nmAu NPs particles in the PBS solution containing PFD, whereas largeaggregates were observed for Au NPs with a size of 15 or 20 nm inthe presence of the chaperone. These results also suggest that thestabilization of Au NPs is achieved by a size-specific interaction be-tween PFD and Au NPs. The diameter size of Au NPs (d = 10 nm)was measured by dynamic light scattering (DLS) both in the ab-sence or presence of PFD. In the first case, the diameter of Au NPwas found to be 9.4 ± 7.0 nm, whereas in the presence of PFD,two peaks of 15.3 ± 5.1 and 132 ± 86.6 nm were observed (see Fig-ure S5). The first of these two values supports the formation of acomplex between Au NP (10 nm diameter) and PFD (�7 nm inheight), whereas the second value may reflect the aggregation ofAu NPs. We sought to confirm by EM the formation of a complexbetween Au NP and PFD. Therefore, Au NP particles were stainedin the absence or presence of PFD (Figure 4, a field of PFD particlescan be seen in Figure S6). The microscopy obtained confirmed notonly the aggregation of Au NP particles (Figure 4A) and the disag-gregating effect exerted by the PFD molecules (Figure 4B) but alsorevealed that this effect takes place by the PFD molecules sur-rounding the Au NP particles (Figure 4B and C), thus preventingtheir interaction and subsequent aggregation.

Our results indicate that PFD is an effective inhibitor of salt-in-duced Au NP aggregation. In previous studies, biomolecules conju-gated to Au NPs were usually prepared by formation of an Au–Sbond between biomolecule thiol groups and the Au surface. How-ever, the chaperone PFD used in this study does not possess anycysteine residues. Therefore, other interactions must take place

for the binding of PFD to Au NPs. Since it has been shown by EMthat PFD interacts with unfolded proteins through hydrophobicresidues located at the tips of tentacles [23], it was thought thatPFD could bind Au NPs by the same mechanism. Our results (Figure4B and C) show that the tips of PFD tentacles interact with Au NPsin a manner similar to that interaction with denatured protein as ithas been shown for different protein such as LYS, GFP or conalbu-min [23], which strengthens the notion that hydrophobic interac-tions are important in PFD-Au NP interactions. Since theinteraction was also achieved under high salt concentration condi-tions, electrostatic interactions are not important in the interactionbetween PFD and Au NPs.

The size-selective recognition of Au NPs by PFD is likely due tothe size of the hydrophobic area formed by the PFD tentacles. Thisnotion is supported by the structure of PFD, which comprises sixlong tentacles encompassing approximately 10 nm, This lengthhas been determined by different techniques such as X-ray crystal-lography in the case of this chaperone [22] or the PFD from thehomologous archaobacterium M thermoautotrophicum [21], andhas been confirmed by EM and image processing in the case ofthe PFD used in this study [23] or the human PFD (homologous

Figure 4. Negative staining TEM of Au NP particles in the absence or presence of PFD. (a) Au NP particles. (b) Au NP particles in the presence of PFD molecules. (c) Amagnification of the complex between Au NP particles and PFD molecules. Arrows point to free PFD molecules. Bar indicates 50 nm.

M. Sakono et al. / Chemical Physics Letters 501 (2010) 108–112 111

to the archaeal one) [25]. An EM study has also shown that PFD caninteract with unfolded proteins of different sizes, using differentnumber of tentacles in a manner that is proportional to the sizeof the denatured protein (i.e. the larger the protein is, the moretentacles are involved in the interaction) so that in the case of un-folded conalbumin (a large protein of 75 kDa), all six tentacles areinvolved in the interaction [23]. We believe this is the case for Au

AuNPs (<10 nm)

in PBS

Aggregation

in PBS containing PFD

PFD

Dispersion

AuNPs (>10 nm)

Weak interaction

in PBS containing PFD

Stronginteraction

Aggregation

A

B

Figure 5. Model of interaction of PFD with the Au NP particles. (A) In the case of theAu NP particles of 10 nm or smaller, the PFD hydrophobic area encompassed by thetips of the six tentacles is capable of embracing the surface of the Au NP particle andtherefore of inhibiting its aggregation. (B) In the case of Au NP particles larger than10 nm, the PFD tentacles cannot properly embrace the particles and therefore theinteraction between PFD and Au NP does not take place.

NP particles of 10 nm size and smaller, whereas Au NP particleslarger than the 10 nm length of the PFD substrate-interacting areacannot be hold by PFD and therefore is not protected by the chap-erone. A schematic illustration of the recognition of Au NP by PFDis shown in Figure 5. PFD can interact tightly with Au NP particlesof 10 nm or smaller, thereby inhibiting salt-induced Au NP aggre-gation (Figure 5A). On the other hand, the inability of the PFD mol-ecules to interact with Au NP particles larger than 10 nm andinhibit their aggregation can be accounted for by the inability ofPFD tentacles to embrace the particle surface and therefore prop-erly interact with the Au NP particles (Figure 5B).

4. Conclusions

In conclusion, we report for the first time the formation of acomplex between a metal colloid and a molecular chaperone. Theadvantages of using PFD include (1) tight interactions with AuNPs in spite of the absence of a thiol group, and (2) an interactionbetween PFD and Au NPs that is size-specific. By utilizing theseproperties, PFD can be used as a bridge protein for the generationof Au NPs with new functions. PFD is also expected to be an effec-tive additive for the production of metal colloids from the view-point of size adjustment and dispersion stability of the metalparticles.

Acknowledgement

This work was supported by the Japan Science TechnologyAgency (PRESTO program). This work was also supported by grantS2009MAT-1507 from the Madrid Regional Government (to JMV).SD is a recipient of ‘La Caixa’ foundation fellowship.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.cplett.2010.10.035.

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