NANO LETTERS
Mechanism Underlying Specificity of Proteins Targeting Inorganic Materials
2006 Vol. 6, No. 3 515-519
Tomohiro Hayashi,*,†,‡,§ Ken-Ichi Sano,|,⊥ Kiyotaka Shiba,|,⊥ Yoshikazu Kumashiro,†,‡ Kenji Iwahori,⊥ Ichiro Yamashita,⊥,# and Masahiko Hara†,‡ Department of Electronic Chemistry, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8502, Japan, Local Spatio-Temporal Functions Laboratory, Frontier Research System, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan, Department of Protein Engineering, Cancer Institute, Japanese Foundation for Cancer Research, and CREST, JST, 3-10-6 Ariake, Koto-ku, Tokyo 135-8550, Japan, Graduate School of Materials Science, Nara Institute of Science and Technology, and CREST, JST, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan, and AdVanced Technology Research Laboratories, Matsushita Electric Industrial Co., Ltd., 3-4 Hikari-dai, Seika, Kyoto 619-0237, Japan Received January 10, 2006; Revised Manuscript Received February 8, 2006
ABSTRACT Adhesion force analysis using atomic force microscopy clearly revealed for the first time the mechanism underlying the specific binding between a titanium surface and ferritin possessing the sequence of Ti-binding peptide in its N-terminal domain. Our results proved that the specific binding is due to double electrostatic bonds between charged residue and surface groups of the substrate. Furthermore, it is also demonstrated that the accretion of surfactant reduces nonspecific interactions, dramatically enhancing the selectivity and specificity of Tibinding peptide.
Interfaces between protein molecules and inorganic materials are now among one of the hottest research topics in various fields, such as biomedicine, biochemistry, biophysics, and even industry.1 In particular, recent progress in combinatorial biology (e.g., the phage display method and other related artificial evolution techniques) enables us to acquire amino acid sequences possessing specific affinities to their target inorganic materials (usually denoted as aptamers or binders), ranging over metals,2-5 semiconductor materials,6-12 carbon nanotubes,13 nanohorns,14 and fullerenes.15 Consequently, the aptamers will provide new approaches to construct novel interfaces of biomolecules and inorganic materials. Until * Corresponding author. E-mail:
[email protected]. † Department of Electronic Chemistry, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology. ‡ Local Spatio-Temporal Functions Laboratory, Frontier Research System, RIKEN (The Institute of Physical and Chemical Research). § Present address: National Metrology Institute of Japan, The National Institute of Advanced Industrial Science and Technology (AIST). | Department of Protein Engineering, Cancer Institute, Japanese Foundation for Cancer Research, and CREST, JST. ⊥ Graduate School of Materials Science, Nara Institute of Science and Technology, and CREST, JST. # Advanced Technology Research Laboratories, Matsushita Electric Industrial Co., Ltd. 10.1021/nl060050n CCC: $33.50 Published on Web 02/18/2006
© 2006 American Chemical Society
now, to place the biomolecules on solid substrates, the solid surface has been modified with a self-assembled monolayer (SAM), lipid bilayer, and plasma surface treatments. There are numerous examples, and one of them was performed by the authors to fabricate a two-dimensional ordered array of cage-shaped proteins carrying inorganic nanodots in the cavity by modifying the silicon substrate surface with a hydrophobic organic compound layer.16,17 Aptamers, however, will change this situation. It can be realized by aptamers that biomolecules with aptamer are anchored directly on the inorganic substrate surface with specific affinity. Biomolecules can be aligned and placed on the prefabricated inorganic patterns via aptamers, which has been long desired by researchers in the fields of bioelectronics, biosensors, micro total analysis systems (TAS), biochemistry, drug delivery systems, implantings, and so on. However, several important issues remain to be unraveled relating aptamers: What kind of force is responsible for the specific binding? What can/cannot be recognized by the aptamer? To answer these questions, first of all, we require a thorough understanding of the mechanism underlying the specific binding between the aptamers and targets. Despite
the number of works concerning the peptide recognizing inorganic materials, few have discussed the mechanism of binding between aptamers and target inorganic materials. In this work, we concentrate our intention on titanium, which is known for its excellent corrosion resistance and is widely used in many applications, such as implanting materials, electrodes, coatings, etc., as a target material. There has been, so far, only one paper which studied the binding mechanism between a Ti-binding peptide (hereafter denoted as minTBP-1) and Ti surfaces.3 In their work, the authors identified the essential amino acid residues for the specific binding by monitoring the effect of the substitution of each residue in the sequence with an alanine residue on the specific binding and proposed a binding model. The difficulty in the elucidation of the binding mechanism stems largely from the following. First, surfaces of inorganic materials are flat, quite unlike the case for biomaterials as targets. Moreover, several different sequences frequently show specific affinity toward the same target material. With the mere knowledge of the sequence, there are serious obstacles to understanding the binding mechanism. A second difficulty is that, in a water environment, the interaction force between two objects is a blend of forces with different origins, such as electrostatic interaction between charged groups, van der Waals interaction, and water-mediated force. Therefore, the task of clarifying which force is responsible or dominant in the specific interaction is perplexing. These difficulties point up the importance of the direct measurement of force operating between target-specific proteins and their target materials. In this work, we employed atomic force microscopy (AFM) to measure the proteinsubstrate interaction, especially the strength of the adhesion between them. Our interest was placed on the ferritin molecules whose N-terminal domain is endowed with minTBP-1,3,18 which exhibits strong affinity with Ti, Si, and Ag surface and not with Au, Cr, Pt, Sn, Zn, Cu, and Fe. By analyzing the adhesion force against various substrates such as Au(111), Si, Ti, SAMs of alkanethiol with different terminal groups, and highly oriented pyrolytic graphite (HOPG), we characterize the specificity of minTBP-1 after fusing with ferritin and the mechanism of the specific binding and discuss the limit of its specificity and selectivity and the effect of the accretion of surfactant. We used two kinds of ferritins in this work. One is recombinant ferritin composed only of the L-type subunit of horse spleen ferritin (hereafter denoted as ∆1-LF). The other is the Ti-binding ferritin (minT1-LF), which bears the hexapeptide sequence of the minTBP-1 (Arg-Lys-Leu-ProAsp-Ala) added at the N-terminus of each ∆1-LF subunit (Figure 1a).19 The sequences in their N-terminal domain are summarized in Table 1. All ferritins were stored in TrisHCl buffer solution (about 50 mM, pH 8.0) until use. Six kinds of substrates were used in this work: Au(111) on a mica substrate (thickness of about 100 nm), a SAM of 11mercapto-1-undecanol (MUOH) and 1-octadecanethiol (ODT) deposited on the Au(111) substrates, Si(100) (p-type, with a 3.5-nm-thick thermally oxidized layer and a root-meansquare (rms) substrate roughness of about 0.3 nm), titanium 516
Figure 1. (a) A picture of a ∆1-LF molecule from the side of the 3-fold symmetric channel. Atoms in the N-terminal domain are represented as van der Waals spheres. In the two-dimensional crystal of ferritin molecules forming the heterobilayer with PBLH, ferritin molecules seem to be oriented with the 3-fold symmetry channels up as shown in this figure. (b) Force as a function of piezomovement on approach and receding for the system of a silicon substrate and Si3N4 AFM tip covered with a heterobilayer of PBLH and minT1-LF in pure water: (1) on approach, attraction is observed; (2) the tip and surface are in hard-wall contact; (3) as the piezo movement is reversed, the tip moves through zero force and is caught in an adhesive well; (4) the tip and surface are out of contact; (5) the tip recedes further. Table 1. Sequences in the N-Terminal Domain
a
ferritin
sequence
∆1-LF minT1-LF
SSQIRQNYST Ma RKLPDA SSQIRQNYST
“M” represents a methionine as a translation start point.
surface evaporated on a glass (thickness of about 200 nm and the rms substrate roughness of about 0.6 nm), and atomically flat HOPG substrates. The AFM system used in this study was the commercially available NanoScope IV with the PicoForce unit that has a closed-loop feed back system for the z direction (Veeco, Inc., Santa Barbara, CA). The radius of the tip curvature was about Nano Lett., Vol. 6, No. 3, 2006
Table 2. Chemical Properties of the Substrate Used in This Work
substrate Au(111) MUOH Si Ti HOPG
Figure 2. The inset shows the adhesion force against Ti, HOPG substrates in pure water. Error bars are standard deviations. We also employed a gold-supported SAM of 1-octadecanethiol (ODT), whose water contact angle is about 110-115°, as a substrate. In this case, from the adhesion force, we found the heterobilayer of ferritin and PBLH was destroyed, because the adhesion between ferritin and the SAM was too strong. The main figure shows adhesion force against Au(111) evaporated on mica, a selfassembled monolayer of 11-mercapto-1-undecanol (MUOH) on an Au(111) substrate, and Si and Ti in pure water.
50 nm, and the spring constant of the lever was about 0.01 N/m. For each cantilever, its spring constant was calibrated by measuring the thermal fluctuations.20 The probes were cleaned by UV-ozone exposure for 15 min just prior to the experiments to remove organic contaminants on the tip surface. Ferritin molecules were immobilized onto an AFM tip made of silicon nitride (Si3N4) using poly[(1-benzyl)-Lhistidine] (PBLH) following the method developed by Furuno et al. with slight modification.21-24 All force-distance curve measurements were performed in an aqueous environment. The substrate and AFM tip were mounted into the AFM apparatus, then solution was injected into a fluid cell. Exposure of the solution to air was avoided as much as possible. After the injection, the system was allowed to equilibrate for 20 min. The pH value of solution after normal measurement was around 5.8 due to dissolved carbon dioxide. We discuss the adhesion strength between ferritin and substrates by comparing the forces required to detach the tip from the substrate as shown in Figure 1b. For all measurements, the pushing force after the contact and the loading rate of the tip were fixed at 250 pN and 200 nm/s, respectively. We measured the force-distance curves at at least 10 positions on the substrate, and in total more than 60 curves were averaged for each system. It is noteworthy that we rarely observed the multistep detach of tip from the surface, indicating that the bonds between the tip and surface in most cases were ruptured simultaneously. Figure 2 presents the adhesion force between the ferritins and hydrophilic substrates. The adhesion forces between minT1-LF and the substrate demonstrates the specific affinity of minT1-LF with Ti substrates compared with Si and MUOH SAM, indicating that minT1-LF can “recognize” titanium among the hydrophilic surfaces. It is clearly seen that the adhesion force strongly depends on the sequence of N-terminal region in cases of Si and Ti substrates. On the Nano Lett., Vol. 6, No. 3, 2006
isoelectric point (IEP)
surface groups
4 -OH 2.6-3.031 -O-, -OH -O-, -OH2+, -OH 5.5-6.032 positively charged33
water contact angle (deg) >20 25-30 >20 >20 85
other hand, in the cases of the substrates of Au(111) and MUOH, such dependence is practically no longer observed. As summarized in Table 2, silanol groups of the oxide layer of Si substrates are deprotonated in pure water. Unlike the case of Si, the -OH groups of the oxide surface of Ti are protonated and deprotonated and -O- and OH2+ groups coexist in pure water.25 On the other hand, Au(111) and MUOH surfaces possess no charged groups on the substrate surface at this condition. From the above facts, it can be concluded that the surface charges play an important role in the specific binding between the minTBP-1 and Ti. Sano and Shiba proposed the binding model in which positively charged arginine and negatively charged aspartic acid residues are bound to -O- and -OH2+ surface groups, respectively.3 As seen from our results, the averaged adhesion force between minT1-LF and Si is approximately half of that between minT1-LF and Ti, suggesting that the abovementioned double electrostatic binding is responsible for the specific binding between minTBP-1 and Ti and that only single electrostatic bond (arginine and -O- group) can be formed for each minTBP-1 chain. In addition, we also measured the adhesion force in solution containing salt and found there is little effect on the adhesion force, indicating ions in the solution do not play an important role for the specific binding between Ti and minT1-LF (see Supporting Information). As clearly seen from the inset of Figure 2 and Table 2, the adhesion force depends strongly on the hydrophobicity or hydrophilicity of the surface; i.e., strong adhesion was observed between ferritin and hydrophobic substrates, in agreement with a previous report on different proteins.26 It was experimentally confirmed that there exits a stable hydration layer at the interface between bulk water and the hydrophilic surface.27 Similarly, protein molecules also possess a stable hydration shell surrounding them.28 When a ferritin molecule approachs a hydrophilic surface, these two hydration layers prevent direct contact between ferritin and the surface, resulting in weak adhesion. In this case, only N-terminal domains, which protrude from the spherical body of the ferritin surface, can interact strongly with the surface groups of the substrate. By contrast, hydrophobic surfaces, such as an HOPG, bear no hydration layer and even may destroy the hydration shell of the ferritin molecule. As a result, the surface allows the direct contact between the surface and ferritin, inducing the strong adhesion force due to van der Waals attraction. In fact, the adhesion between ferritin and ODT was too strong to destroy the binding 517
Figure 3. Adhesion force against Au(111) evaporated on mica, a self-assembled monolayer of 11-mercapto-1-undecanol (MUOH) on an Au(111) substrate, and Si and Ti in water containing TWEEN20 surfactant at a concentration of 0.5 wt %.
between PBLH and ferritin (see Supporting Information). In this instance, the adhesion between ferritin and hydrophobic surfaces does not depend on the sequence in the N-terminal domain, because the ferritin shell adheres to hydrophobic substrates directly, which is so strong that the adherent force difference originating from the sequences gives little effect on the final adhesion force. On the basis of our results displayed above, it is clear that minT1-LF however cannot distinguish its target in the presence of hydrophobic objects, due to the overwhelming adhesion force. In biological research, the usual way to sample only specific binding is to add surfactant to the solution.29 In fact, for the isolation of TBP from the peptide phage library, poly(oxyethylene) sorbitan monolaurate (TWEEN20) (Sigma Aldrich, St. Louis, MO) surfactant and the blocking with bovine serum albumin (BSA) were employed. Figure 3 shows the adhesion force between ferritins and substrates in water containing TWEEN20 at a concentration of 0.5 wt %. Although the adhesion force decreased dramatically in solution with the surfactant, minT1LF show the highest adhesion force to the Ti substrate, and this adhesion overcomes that to HOPG, demonstrating that nonspecific interactions are suppressed and the specificity and selectivity of the minT1-LF are enhanced due to the accretion of the surfactant. To understand the role of TWEEN20, we performed the QCM (Q-Sense AB, Vaestra Froelunda, Sweden) measurements to observe the adsorption behavior of TWEEN20 surfactant molecules onto the substrates (gold, MUOH, ODT, Si, and Ti) as shown in Figure 4. For all substrates, it is confirmed that the surfactant molecules do adsorb on to the surface, although the amount of the adsorption depends on the substrate. We expect the surfactant molecules would surround the substrates and ferritin molecules and this would reduce the nonspecific interactions such as hydrogen bonding and hydrophobic interaction. As clearly seen in Figure 4, the Ti substrate adsorbs fewer TWEEN20 molecules than other substrates. This may result in the smaller decrease in 518
Figure 4. QCM measurements of the adsorption TWEEN20 onto gold, gold-supported MUOH, ODT, Si, and Ti substrates The shift in the resonant frequency is plotted as a function of time after the injection of the TWEEN20 solution. First the QCM sensors were stabilized in deionized water and water containing TWEEN20 at a concentration of 0.5 wt %. After the adsorption curve was stabilized, deionized water was injected for rinsing. On the basis of a simple Sauerbrey equation, the change of 1 Hz in the resonance frequency corresponds to 17.7 ng/cm2. The bars beside the right axis represent the shift of the resonant frequency in equilibrium just before rinsing.
adhesion force compared with other cases. Unfortunately, we could not prepare the substrates that reproduce the HOPG surface for QCM measurements. However, guessing from hydrophobicity of HOPG (Table 2) and the result of the hydrophobic ODT that adsorbed TWEEN20 molecules, HOPG would adsorb TWEEN20 molecules, resulting in the decrease in the adhesion force. Although the adsorption behavior of TWEEN20 molecules onto the ferritin molecule is not clear, our result clearly demonstrates that the surfactant dramatically enhances the specificity of minT1-LF to Ti, as was similarly proved in the experiment performed by Kirimura et al. who succeeded in the selective adsorption of minT1-LF onto the islands of Ti evaporated on a silicon substrate.30 In this work we elucidated, for the first time, the mechanism underlying the specific binding between a targetspecific protein and the target in a direct way. First, we showed that minTBP-1 exhibited the specificity even after being introduced into the N-terminal domain of each subunit of ferritin molecules. Our results also revealed that the sequence of RKLPDA in the minT1-LF’s N-terminal domains is strongly bound to charges originating from the protonation and deprotonation of the surface groups of a Ti substrate, in the same manner proposed by Sano and Shiba.3 We also demonstrated that the hydrophilicity or hydrophobicity is an important factor to govern the adhesion force between ferritin and the substrate. Against a hydrophobic surface, the strength of the adhesion exceeds the strength of the specific binding between minT1-LF and Ti, indicating that minT1-LF might not distinguish the target when the target is mixed with hydrophobic objects. To avoid such a situation, we proposed that the selectivity and specificity will be enhanced by adding surfactant to the solution; i.e., the surfactant will surely be expected to work as a restraining Nano Lett., Vol. 6, No. 3, 2006
factor that suppresses nonspecific bindings and nonspecific biological reactions. Acknowledgment. Dr. Mishima is gratefully acknowledged for providing us with the PDB file of the ∆1-LF molecule. This study is supported by the Leading Project of the Ministry of Education, Culture, Sports, Science and Technology. We thank Prof. Kaoru Tamada for kind and helpful discussions. Supporting Information Available: Description of the sample preparation, discussion of stability of the heterobilayer of PBLH and ferritin and effect of solution conditions on the adhesion force, and figures showing adhesion forces measured in Tris-HCl buffer with NaCl and NaCl containing TWEEN20. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) (2) (3) (4) (5) (6) (7) (8) (9) (10)
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