Journal of Colloid and Interface Science 442 (2015) 82–88
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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Two-dimensional self-assembly of amphiphilic peptides; adsorption-induced secondary structural transition on hydrophilic substrate Masayoshi Tanaka a,⇑, Souhei Abiko b, Takahiro Himeiwa b, Takatoshi Kinoshita b a b
Department of Applied Chemistry, Meijo University, 1-501 Shiogamaguchi, Tempaku-ku, Nagoya 468-8502, Japan Department of Frontier Materials, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan
a r t i c l e
i n f o
Article history: Received 3 September 2014 Accepted 7 November 2014 Available online 14 November 2014 Keywords: 2D self-assembly Solid/water interface Secondary structural transition
a b s t r a c t Adsorption of sequential amphiphilic peptides on solid substrates triggered the spontaneous construction of nanoscaled architecture. An amphiphilic peptide designed with a cationic amino acid as a hydrophilic residue turned an anionic mica substrate into a water-repellent surface, simply by adsorbing it on the substrate surface. In contrast, an amphiphilic peptide designed with an anionic amino-acid residue formed a precisely controlled fiber array comprising a b-sheet fiber monolayer at the anionic substrate/water interface. This phenomenon was based on the secondary structural transition from random-coil to b-sheet, which occurred specifically when amphiphilic peptide adsorbed on the substrate surface. Such surface-specific nonorder/order transition was implemented by exploiting the strength of adsorption between the peptide and the substrate. A strategic design exploiting weak bonding such as hydrophobic interactions is essential for constructing precisely controlled nano-architectures in two dimensions. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Construction of two-dimensional (2D) planar structures with controlled orientation and functional-group location is of great importance in surface modification and functionalization. Precisely controlled thin layers are attractive as templates for biomaterials [1,2], nanoparticle arrays [3–5], and bio-mineralization [6–8]. Among the studied preparation methods, adsorption-based strategies are some of the most practical because of their simplicity. Amphiphilic peptides are valuable candidates for fabricating regulated 2D architectures because of their potential in synthesizing tailor-made designs by selecting amino-acid residues with the desired hydrophobic and ionic groups [9–13]. The formation of secondary structures such as a-helices, b-sheets, and random-coils is another characteristic feature of peptides. In particular, b-sheetforming peptides can build relatively long controlled structures such as nanoscale fibrous objects owing to the intermolecular hydrogen bonding among the amide groups of the peptide backbone. The orientation of b-sheet peptides is also controlled by the side chains of the neighboring amino acid residues facing opposite sides of the backbone. Thus, b-sheet-forming peptides
⇑ Corresponding author. Fax: +81 52 838 1179. E-mail address: [email protected] (M. Tanaka). http://dx.doi.org/10.1016/j.jcis.2014.11.021 0021-9797/Ó 2014 Elsevier Inc. All rights reserved.
are one of the most fascinating adsorbents, in which the orientation and location of the functional groups can be precisely controlled. Amphiphilic peptides with a simple sequence of alternating hydrophobic and hydrophilic residues are one of the most promising candidates to fabricate b-sheet structures [14–16]. In fact, amphiphilic peptides show 2D fiber arrays that are regulated on the nanometer scale [17–21]. In these studies, the strategy to fabricate a 2D-regulated planar structure is based on adsorption of the ordered structure already formed in the solution as a 3D environment. There should be a marked attractive force between the adsorbent and the substrate to stabilize the adsorbed state because of the small energy gap between the ordered structures in the 3D and 2D environments. We have focused on the disorder/order transition of the peptide adsorbent. If such structural transition occurs specifically at the solid/water interface, it is expected to provide a novel approach to fabricate 2D-regulated planar structure triggered with the adsorption. A peptide adopting a random-coil conformation as a typical disordered state is one of the most intriguing adsorbents. The random-coil that is incomplete hydrogen bonding between amide backbones can readily form another ordered structure with a small triggers. We have previously reported that a nanoscaled fiber array is spontaneously formed by the adsorption of the poly(ethylene glycol) conjugated anionic peptide on a mica substrate [22,23]. It was suggested that the poly(ethylene glycol) segment critically worked as an anchor to
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stabilize the adsorption of the anionic peptide moiety. Thus, potentiality as an adsorbent and the essential interfacial behavior of the amphiphilic peptide substance are still unclear. In this study, anionic and cationic peptide amphiphiles comprising a series of ionic hydrophilic/hydrophobic amino-acid sequences were designed. We investigated the 2D self-assembly of these peptides on the surface of a hydrophilic substrate by immersing the substrate in the peptide solution. 2. Materials and methods 2.1. Synthesis The cationic amphiphilic peptide, LK, comprises eight sequential units of alternating leucine (L) and lysine (K); hence this peptide can be denoted as Ac-(LK)8-NH2. The anionic amphiphilic peptide, LE, was designed using glutamic acid (E) as an anionic residue instead of K, Ac-(LE)8-NH2. The peptides, with 16 amino-acid residues, were prepared by a standard solid-phase synthetic strategy by following a standard Fmoc-protection protocol. The N-terminus of the resulting peptides was acetylated. Cleavage and deprotection with trifluoroacetic acid yielded the peptide moiety. The molecular weight of the peptides was estimated by MALDI-TOF MS. LK (MALDI-TOF MS); [M + H]+: observed, 1989.9; calculated, 1989.6, LE (MALDI-TOF MS); [M + Na]+: observed, 2084.1; calculated, 2084.1. A stock solution of LK was prepared by dissolving it in pure water to yield a concentration of 5.0 10 4 M. For performing immersion experiments, the pH values of the aqueous solutions were fixed according to pKa of the polymers designed the ionic amino acid residues. The pKa values of the lysine and glutamic acid designed polymer are 9.4–10.5 and 4.4–4.9, respectively [24–26]. The aqueous solutions with pH values 6.0 and 11.0 were prepared by diluting the stock solution with pure water and 0.01 M sodium hydroxide (NaOH) solution, respectively. The final concentration of these solutions was 5.0 10 5 M. A stock solution of LE was prepared by dissolving it in 0.01 M NaOH to yield a concentration of 5.0 10 4 M. Another aqueous solution for performing the immersion experiments was prepared by diluting the stock solution with 0.01 M NaOH solution to achieve a final concentration of 5.0 10 5 M and pH 12.0. An acidic solution of the peptide was prepared by adding 0.02 M hydrochloric acid to the stock solution to adjust the pH to 3.0. The final concentration of the acid solution was 5.0 10 5 M. 2.2. Adsorption of peptides on substrate Prior to use, a mica substrate was freshly cleaved, and quartz and calcium fluoride (CaF2) substrates were washed with HNO3 and an alkaline detergent (SCAT 100X-U), respectively. The washed substrates were rinsed with pure water and dried under atmospheric conditions. The peptides were adsorbed on the substrate by immersing the substrate in the aqueous peptide solutions at 25 °C. The substrate obtained by varying the immersion duration was rinsed by dipping it in pure water and dried under atmospheric conditions. 2.3. Characterization Circular dichroism (CD) spectra of the peptides were recorded on a Jasco J-820 K spectropolarimeter (JASCO Corp., Jpn) under a nitrogen atmosphere. The CD measurements of the adsorbed state on the quartz substrate were performed over the range 190–260 nm at room temperature. Four quartz substrates were layered to enhance the intensity of ellipticity.
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Fourier transform infrared (FT-IR) spectra of the peptides were measured using an FT-IR spectrometer SPECTRUM 2000 (Perkin Elmer). The FT-IR measurements were carried out over the range 1000–4000 cm 1. Five CaF2 substrates were layered to enhance the intensity of the absorbance. Atomic force microscopy (AFM) measurements were made in air using Digital Instruments Multimode Nanoscope IV (Bruker AXS) in tapping mode. A silicon cantilever (Nanoworld, NCH-10T; length, 125 lm; tip radius, Rc = 8 nm) was used for imaging. The size of the fiber is an average of more than 20 points of the fibers. The standard deviation of LE and LK were 6.4% and 11.6%, respectively. A literature-based calculation method was used to estimate the plausible size of a flat shaped structure such as a b-sheet peptide fiber while considering the tip convolution effect in AFM imaging [27]. Contact angle measurements were made to determine changes in the hydrophilicity of the mica substrate on their modification by the peptides using an optical contact angle meter (Kyowa Interface Science Co., Ltd., Drop Master 500). A drop of pure water (2 ll) was manually deposited on the peptide-modified surface of the mica substrate. An average contact angle value was obtained by measuring the same sample at five different positions. 3. Results and discussion 3.1. Configuration of peptide adsorbents in aqueous solutions The secondary structure of a peptide containing ionic amino acid residues generally depends on the solution pH. According to the pKa of the amino group [25,26], the side chain of the lysinecontaining peptide is protonated (LK+) at pH 6.0 and deprotonated (LK) at pH 11.0. The CD spectrum of the aqueous solution of LK+ at pH 6.0 showed a negative peak at 195 nm, which is typical for a random-coil structure (Fig. 1A) [28]. When the LK solution was prepared at pH 11.0, the CD spectrum showed a negative peak at 215 nm and a positive peak at 195 nm (Fig. 1A). LK formed a typical b-sheet conformation when the side chain was deprotonated [28]. The pKa values of the carboxylic acid [24,25] show that the side chain of the glutamic acid-containing peptide is protonated (LE) at pH 3.0 and deprotonated (LE ) at pH 12.0. The CD spectrum of the aqueous solution of LE at pH 3.0 showed a negative peak at 215 nm and a positive peak at 195 nm, indicating a typical b-sheet conformation (Fig. 1B). The CD spectrum of LE at pH 12.0 showed a negative peak at 198 nm, which is typical for a random-coil structure (Fig. 1B). Both peptides adopted a highly dispersed configuration as random-coils in aqueous solution when the side chain was charged. 3.2. Adsorption of an amphiphilic peptide containing cationic amino acid residues The 2D self-assembly of LK+, which is designed with a cationic amino acid as a hydrophilic residue, was investigated. A mica substrate was immersed in the solution of LK+ with a random-coil conformation. The AFM image of the mica substrate immersed for 18 h as a typical sample showed fiber-like objects, 23–35-nm width and 5-nm height (Fig. 2A). When a mica substrate was immersed in an aqueous solution of LK at pH 11.0, in which the peptide adopted a b-sheet conformation, 10-nm-wide fibrous objects were observed on the mica surface (Fig. 2B). The width and height of these objects were estimated to be 13.6 and 1.4 nm, respectively. Considering the radius of curvature of the AFM probe, the width was calculated to be 4.6 nm. This value agreed with the expected length of a b-sheet peptide with 16 residues, i.e., 5.6 nm. The height of the fibrous object was twice as high as the monolayer of the peptide with the b-sheet conforma-
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Fig. 1. CD spectra of (A) aqueous solution of (a) LK+ at pH 6.0 and (b) LK at pH 11.0. (B) Aqueous solution of (a) LE at 3.0 and (b) LE at pH 12.0.
Fig. 2. AFM images of adsorbed (A) LK+ at pH 6.0 and (B) LK at pH 11.0 on mica substrate; immersion duration: 18 h; height scale: 10 nm.
tion. Amphiphilic peptides designed with alternating hydrophilic/ hydrophobic amino acids often form bilayered fibrous objects with inward-facing hydrophobic residues. The observed fiber on the mica surface was attributed to the adsorption of the b-sheet peptide fiber formed in the solution. The observed object from LK+ solution was apparently large both in the width and height compared with the b-sheet peptide fiber. It was suggested that the 2D self-assembly according to the adsorption of LK+ was proceeding by complicated manner. The CD spectrum of the adsorbed peptide on the quartz substrates obtained by immersing in the LK+ solution at pH 6.0 showed negative peak around 211 nm (Fig. 3A). In the case of the LK peptide adsorbed quartz substrates, a negative peak was observed at 215 nm, which is typical for a b-sheet conformation. The negative peak obtained from the LK+ solution is slightly shifted to a shorter wavelength from the typical peak of the b-sheet peptide.
Such a peak shift was previously proposed for the mixed randomcoil and b-sheet conformations [28]. The FT-IR spectra of the peptide layers obtained from the LK and LK+ solution were observed (Fig. 3B). The layer obtained from LK solution where the peptide adopted b-sheet conformation showed 1629 cm 1 in the amide I region (solid line in Fig. 3B) [29]. This peak typically assigned to b-sheet conformation supported the LK layer was composed with b-sheet fibers. On the other hand, the LK+ layer showed the peaks at 1645 cm 1 attributed to random-coil (dashed line in Fig. 3B). A small peak at 1626 cm 1 was also observed in this spectrum. This peak corresponded to b-sheet conformation. The observed amide I bands indicated that the planar structure obtained by LK+ adsorption comprised a mixture of b-sheet and random-coil structures. In the case of the combination of oppositely charged cationic adsorbent and anionic substrate, the ionic attractive force dominated the adsorption on the substrate surface. The cationic
Fig. 3. (A) CD spectra of (a) LK+ and (b) LK adsorbed on quartz substrates. (B) FT-IR spectra of (a) LK+ and (b) LK adsorbed on CaF2 substrates.
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residues already exposed to the outer phase in the random-coil configuration readily contact the surface of the substrate and stabilize the adsorbed state as a random-coil. As the ionic interaction progresses, the hydrophobic residues containing eight residues in the 16-mer peptide are inevitably exposed to the outer phase, though the exposure of the hydrophobic residues is energetically unfavorable in aqueous medium. Then, a second layer is formed by the adsorption of additional LK+ molecules from the bulk solution to cover the exposed hydrophobic phase. The constructed large fibrous objects formed by LK+ adsorption can be explained by the complex self-assembly, which involves both the adsorption of the random-coil and the secondary structural transition into the b-sheet structures. In this multidimensional propagation, a secondary structural transition may be induced to form a totally stable planar structure accompanying the topological effect of the peptide self-assembly. It is noteworthy that the obtained LK+ layer on the mica substrate was apparently water-repellent (Fig. 4). The contact angle of the LK+ layer on the mica substrate was 95.2°. This value was much larger than that of the freshly cleaved mica surface (7.0°). An increase in the contact angle was also observed when the immersion duration was short as 1 s. The cationic peptide immediately covered the mica surface and made it water-repellent.
3.3. Adsorption of an amphiphilic peptide containing anionic amino acid residues The 2D self-assembly behavior when LE peptide adopting a random-coil conformation adsorbed on the surface was investigated. The AFM images showed fibrous objects on the surface of the mica substrate (Fig. 5). The width of the fibers was estimated to be 11.9 nm. Considering the radius of curvature of the AFM probe, the estimated width was calculated to be 5.8 nm. This value agreed well with the expected length of a b-sheet peptide with 16 residues, i.e., 5.6 nm. The height of the fibers was 0.6 nm (Fig. 5F), which corresponds to the thickness of a single-layered b-sheet peptide comprising L and E residues adopting an extended state such as a b-sheet conformation. The peptide fibers occupied the surface of the substrate as a function of the immersion time (Fig. 5A–E). This result suggests that the secondary structure of LE dissolved as a random-coil spontaneously transformed into a b-sheet when adsorbed on the mica substrate. The mica substrate was immersed in the peptide solution with pH 3.0; in such a solution, the peptide formed a b-sheet conformation. Fibrous objects were sporadically observed in the AFM images (Fig. 6). Their width and height were estimated to be 14.3 and
Fig. 4. Photograph of the water drops on the LK+ peptide coated mica substrate.
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1.3 nm, respectively. The plausible width value was calculated to be 5.6 nm, which was agreed well with that of the b-sheet fiber comprising 16 residues. The height of the fiber coincided with that of a bilayered object. Like the LK peptide, the LE peptide adopting b-sheet conformation also forms a bilayered fibrous object by facing hydrophobic residues in the solution. The adsorption of the bilayered fiber was very poor compared to the LE adsorption at the same duration of the immersion (Fig. 5E). It suggests that the bilayered structure of the LE peptide with a b-sheet conformation exposing the hydrophilic residues to the outer surface has a low affinity for the hydrophilic mica surface. The CD spectrum of the peptide adsorbed on the quartz substrates immersed in the LE solution at pH 12.0 showed negative and positive peaks at 215 and 195 nm, respectively (Fig. 7A). The adsorbed peptide obtained from the pH 3.0 solution also showed negative and positive peaks at 217 and 195 nm, respectively (Fig. 7A). Both spectra typically indicate a b-sheet conformation. The CD spectra showed that the random-coil/b-sheet transition occurred when the LE peptide adsorbed on the surface of the quartz substrate. The FT-IR spectrum of the peptide layer obtained from the random-coil solution showed peaks at 1542, 1631 and 1698 cm 1 (solid lines in Fig. 7B). These amide I and II bands indicate that the peptide layer adsorbed on the CaF2 substrates is composed of antiparallel b-sheet structures. These bands were also observed for the adsorbed peptide layer obtained from the b-sheet peptide solution at pH 3.0 (Fig. 7B). The results indicate that the peptide in the random-coil state apparently transforms its secondary structure to an antiparallel b-sheet at the solid/water interface. In the FT-IR spectrum obtained from the LE solution, additional peaks were observed at 1578 and 1467 cm 1 (dashed lines in Fig. 7B). The carboxylate salt shows asymmetrical and symmetrical stretching bands around 1570 and 1460 cm 1, respectively [30,31]. The observed peaks, which are peculiar for the peptide layer obtained from the random-coil solution, correspond to the infrared absorption of COO of the glutamic acid side chains. This suggests that b-sheet fibers were formed regardless of anionic repulsion at the interface, and the anionic species derived from the ionized carboxyl side chains were maintained in the 2D self-assembling system. To verify the effect of the ionic species on the 2D self-assembly, an immersion experiment with salt was performed; generally, ionic repulsion is decreased by the addition of salt. A solution of LE with KNO3 (10 equivalents to the anionic residue) was freshly prepared, and the mica substrate was immersed in this solution for 18 h. Nanoscaled fibers were also observed in the AFM images (Fig. 8). Their width and height were in agreement with those of the b-sheet peptide. In the AFM image, the fibrous objects were sporadically layered. The height of the layered region was 1.3 nm, which was equivalent to the height of 2 b-sheet layers. The peptide adsorbed on the substrate first vertically aggregated because of the reduced ionic repulsion, and then transitioned from a random-coil to b-sheet conformation in the first layer of the fibrous object. The orientation of the obtained monolayer of b-sheet fiber was estimated by contact angle measurement. The contact angle of the peptide monolayer obtained from the random-coil solution was estimated to be 15.9°. For comparison, orientation-controlled zpeptide monolayers were prepared conventionally by self-assembly at the air/water interface. When the amphiphilic peptide adopting a b-sheet conformation spreads at the air/water interface, it orients the hydrophobic and hydrophilic residues toward the air and water phases, respectively. The Langmuir–Blodgett (LB) method [32,33] enables the fabrication of the amphiphilic peptide monolayer exposing the hydrophobic residues on a substrate by skimming the peptide monolayer from the water surface. On the other hand, the Langmuir–Schaeffer (LS) method [34,35] enables the fabrication of the amphiphilic peptide monolayer exposing
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Fig. 5. (A–E) AFM images of adsorbed LE at pH 12.0 after 1–18 h; height scale: 5 nm. (F) Section analysis with a line graph of the segment shown in E.
Fig. 6. (A) AFM image of adsorbed LE at pH 3.0 after 18 h; height scale: 5 nm. (B) Section analysis with a line graph of the segment shown in A.
the hydrophilic residues, because the monolayer constructed on the water surface adheres to the horizontally approaching substrate. Both LB and LS films were prepared on mica substrate by spreading LE peptide on the air/water interface. The contact angles of the LB and LS films were estimated to be 42.4° and 16.8°, respectively. The contact angle of the peptide monolayer obtained by the adsorption-based method from the random-coil
solution (15.9°) nearly coincided with that of the LS film. Accordingly, it is conceivable that the orientation of the peptide monolayer constructed by the adsorption of the random-coil was controlled with the hydrophilic residues exposed at the outer surface. Conversely, it indicated that the adsorption of the b-sheet nanofiber was stabilized by the hydrophobic interactions. Stabilization of the random-coil in aqueous solution is achieved
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Fig. 7. (A) CD spectra of (a) LE and (b) LE adsorbed on quartz substrates. (B) FT-IR spectra of (a) LE and (b) LE adsorbed on CaF2 substrates.
Fig. 8. (A) AFM image of adsorbed LE at pH 12.0 with KNO3; height scale: 5 nm. (B) Section analysis with a line graph of the segment shown in A.
Fig. 9. Plausible images of the configuration constructed by the amphiphilic peptide (A) in the aqueous solution adopting a random-coil conformation and (B) on the substrate adopting a b-sheet conformation.
by a configuration that hides the hydrophobic residues within the hydrophilic moieties. However, in the random-coil configuration, the hydrophobic residues that occupy half of the amino-acid residues in the 16-mer peptide are sterically crowded, and hence,
partially exposed to the outer surface. Thus, the random-coil dissolved in the aqueous solution inevitably possesses regions with high surface energy. Owing to the nonhomogeneous surface character of the random-coil, the peptide can adsorb onto the
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substrate by hydrophobic interaction. The peptide then transformed to a b-sheet conformation, when the 2D density of the peptide increased over a critical point as the adsorption proceeded (Fig. 9). In the aqueous solution of LE at pH 3.0, the critical aggregation concentration (CAC) was estimated to be 6.8 10–7 M by fluorescence measurements using pyrene. CD measurements showed that the peptide adopted a b-sheet conformation above the CAC. In contrast, the CAC of LE at pH 12.0 was not detected in the aqueous medium. The peptide showed CD spectra supporting only a random-coil conformation in the soluble concentration regime. The surface of the substrate may offer a self-assembling field where the LE can aggregate above a critical value. Then, the adsorbed LE underwent the secondary structural transition to gain stability by inter-strand hydrogen bonding. Such 2D fibrillization based on the b-sheets of the anionic peptide amphiphile occurred in spite of the ionic repulsion between the anionic moieties on the side chain. Such lateral aggregation at the interface probably occurs upon modulating the competition between the stabilizing effects of the interstrand hydrogen bonding and hydrophobic interactions and the destabilizing effect of the ionic repulsion. When the sequential peptide took an anti-parallel b-sheet conformation, the distances between the neighboring side chains in the hydrophilic face (lch) and peptide strands (lstr) were 0.7 and 0.47 nm, respectively [9,36,37]. The value of lch is comparable to value of the Bjerrum length (LB) [38], at which the potential electric energy of two elementary charges is equal to the thermal energy (kBT). The fact that lstr is shorter than LB suggests that the aggregation of the peptide strands is influenced by the ionic repulsion. For instance, when amphiphiles such as fatty acids and lipids form a lamellar structure or monolayer, the distance between the hydrophilic head groups in the 2D close-packed structure is reported to be 0.45–0.49 nm [39,40]. This means that amphiphiles can aggregate within the LB regardless of the ionic repulsion between the hydrophilic heads in aqueous solution, if there are supplemental stabilizing factors such as hydrophobic interactions. Considering that the distance between the peptide strands adopting a b-sheet conformation is approximately equal to the size of the head groups of the amphiphiles, the dimensions of the aggregated peptide with anionic side chains are reasonable. In the 2D environment, the LE formed a b-sheet conformation supplemented by hydrophobic interactions and inter-strand hydrogen bonding, while it took a random-coil conformation in a 3D environment such as a bulk solution, where it was influenced by hydrophobic interactions and ionic repulsion. 4. Conclusions We have demonstrated the spontaneous construction of a 2D layered architecture by the adsorption of specially designed amphiphilic peptides with ionic residues as hydrophilic moieties. A sequential peptide comprising cationic hydrophilic/hydrophobic amino-acid residues formed a water-repellent planar architecture. The obtained architecture was based on a mixture of random-coil and b-sheet conformations. These results show that the combination of oppositely charged peptide and substrate stabilized a relatively rigid topological structure based on ionic attractive forces. On the other hand, a sequential peptide designed with a series of anionic hydrophilic/hydrophobic amino acid residues underwent a random-coil/b-sheet transition at the substrate/water interface, yielding a nanoscale fiber array. The obtained structure was an orientation-controlled monolayer, in which the hydrophilic residues are exposed at the outer surface. Interestingly, even though both the peptide and substrate are anionic, the adsorption of the peptide actually resulted in a monolayered b-sheet fiber array. This
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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Two-dimensional self-assembly of amphiphilic peptides; adsorption-induced secondary structural transition on hydrophilic substrate Masayoshi Tanaka a,⇑, Souhei Abiko b, Takahiro Himeiwa b, Takatoshi Kinoshita b a b
Department of Applied Chemistry, Meijo University, 1-501 Shiogamaguchi, Tempaku-ku, Nagoya 468-8502, Japan Department of Frontier Materials, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan
a r t i c l e
i n f o
Article history: Received 3 September 2014 Accepted 7 November 2014 Available online 14 November 2014 Keywords: 2D self-assembly Solid/water interface Secondary structural transition
a b s t r a c t Adsorption of sequential amphiphilic peptides on solid substrates triggered the spontaneous construction of nanoscaled architecture. An amphiphilic peptide designed with a cationic amino acid as a hydrophilic residue turned an anionic mica substrate into a water-repellent surface, simply by adsorbing it on the substrate surface. In contrast, an amphiphilic peptide designed with an anionic amino-acid residue formed a precisely controlled fiber array comprising a b-sheet fiber monolayer at the anionic substrate/water interface. This phenomenon was based on the secondary structural transition from random-coil to b-sheet, which occurred specifically when amphiphilic peptide adsorbed on the substrate surface. Such surface-specific nonorder/order transition was implemented by exploiting the strength of adsorption between the peptide and the substrate. A strategic design exploiting weak bonding such as hydrophobic interactions is essential for constructing precisely controlled nano-architectures in two dimensions. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Construction of two-dimensional (2D) planar structures with controlled orientation and functional-group location is of great importance in surface modification and functionalization. Precisely controlled thin layers are attractive as templates for biomaterials [1,2], nanoparticle arrays [3–5], and bio-mineralization [6–8]. Among the studied preparation methods, adsorption-based strategies are some of the most practical because of their simplicity. Amphiphilic peptides are valuable candidates for fabricating regulated 2D architectures because of their potential in synthesizing tailor-made designs by selecting amino-acid residues with the desired hydrophobic and ionic groups [9–13]. The formation of secondary structures such as a-helices, b-sheets, and random-coils is another characteristic feature of peptides. In particular, b-sheetforming peptides can build relatively long controlled structures such as nanoscale fibrous objects owing to the intermolecular hydrogen bonding among the amide groups of the peptide backbone. The orientation of b-sheet peptides is also controlled by the side chains of the neighboring amino acid residues facing opposite sides of the backbone. Thus, b-sheet-forming peptides
⇑ Corresponding author. Fax: +81 52 838 1179. E-mail address: [email protected] (M. Tanaka). http://dx.doi.org/10.1016/j.jcis.2014.11.021 0021-9797/Ó 2014 Elsevier Inc. All rights reserved.
are one of the most fascinating adsorbents, in which the orientation and location of the functional groups can be precisely controlled. Amphiphilic peptides with a simple sequence of alternating hydrophobic and hydrophilic residues are one of the most promising candidates to fabricate b-sheet structures [14–16]. In fact, amphiphilic peptides show 2D fiber arrays that are regulated on the nanometer scale [17–21]. In these studies, the strategy to fabricate a 2D-regulated planar structure is based on adsorption of the ordered structure already formed in the solution as a 3D environment. There should be a marked attractive force between the adsorbent and the substrate to stabilize the adsorbed state because of the small energy gap between the ordered structures in the 3D and 2D environments. We have focused on the disorder/order transition of the peptide adsorbent. If such structural transition occurs specifically at the solid/water interface, it is expected to provide a novel approach to fabricate 2D-regulated planar structure triggered with the adsorption. A peptide adopting a random-coil conformation as a typical disordered state is one of the most intriguing adsorbents. The random-coil that is incomplete hydrogen bonding between amide backbones can readily form another ordered structure with a small triggers. We have previously reported that a nanoscaled fiber array is spontaneously formed by the adsorption of the poly(ethylene glycol) conjugated anionic peptide on a mica substrate [22,23]. It was suggested that the poly(ethylene glycol) segment critically worked as an anchor to
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stabilize the adsorption of the anionic peptide moiety. Thus, potentiality as an adsorbent and the essential interfacial behavior of the amphiphilic peptide substance are still unclear. In this study, anionic and cationic peptide amphiphiles comprising a series of ionic hydrophilic/hydrophobic amino-acid sequences were designed. We investigated the 2D self-assembly of these peptides on the surface of a hydrophilic substrate by immersing the substrate in the peptide solution. 2. Materials and methods 2.1. Synthesis The cationic amphiphilic peptide, LK, comprises eight sequential units of alternating leucine (L) and lysine (K); hence this peptide can be denoted as Ac-(LK)8-NH2. The anionic amphiphilic peptide, LE, was designed using glutamic acid (E) as an anionic residue instead of K, Ac-(LE)8-NH2. The peptides, with 16 amino-acid residues, were prepared by a standard solid-phase synthetic strategy by following a standard Fmoc-protection protocol. The N-terminus of the resulting peptides was acetylated. Cleavage and deprotection with trifluoroacetic acid yielded the peptide moiety. The molecular weight of the peptides was estimated by MALDI-TOF MS. LK (MALDI-TOF MS); [M + H]+: observed, 1989.9; calculated, 1989.6, LE (MALDI-TOF MS); [M + Na]+: observed, 2084.1; calculated, 2084.1. A stock solution of LK was prepared by dissolving it in pure water to yield a concentration of 5.0 10 4 M. For performing immersion experiments, the pH values of the aqueous solutions were fixed according to pKa of the polymers designed the ionic amino acid residues. The pKa values of the lysine and glutamic acid designed polymer are 9.4–10.5 and 4.4–4.9, respectively [24–26]. The aqueous solutions with pH values 6.0 and 11.0 were prepared by diluting the stock solution with pure water and 0.01 M sodium hydroxide (NaOH) solution, respectively. The final concentration of these solutions was 5.0 10 5 M. A stock solution of LE was prepared by dissolving it in 0.01 M NaOH to yield a concentration of 5.0 10 4 M. Another aqueous solution for performing the immersion experiments was prepared by diluting the stock solution with 0.01 M NaOH solution to achieve a final concentration of 5.0 10 5 M and pH 12.0. An acidic solution of the peptide was prepared by adding 0.02 M hydrochloric acid to the stock solution to adjust the pH to 3.0. The final concentration of the acid solution was 5.0 10 5 M. 2.2. Adsorption of peptides on substrate Prior to use, a mica substrate was freshly cleaved, and quartz and calcium fluoride (CaF2) substrates were washed with HNO3 and an alkaline detergent (SCAT 100X-U), respectively. The washed substrates were rinsed with pure water and dried under atmospheric conditions. The peptides were adsorbed on the substrate by immersing the substrate in the aqueous peptide solutions at 25 °C. The substrate obtained by varying the immersion duration was rinsed by dipping it in pure water and dried under atmospheric conditions. 2.3. Characterization Circular dichroism (CD) spectra of the peptides were recorded on a Jasco J-820 K spectropolarimeter (JASCO Corp., Jpn) under a nitrogen atmosphere. The CD measurements of the adsorbed state on the quartz substrate were performed over the range 190–260 nm at room temperature. Four quartz substrates were layered to enhance the intensity of ellipticity.
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Fourier transform infrared (FT-IR) spectra of the peptides were measured using an FT-IR spectrometer SPECTRUM 2000 (Perkin Elmer). The FT-IR measurements were carried out over the range 1000–4000 cm 1. Five CaF2 substrates were layered to enhance the intensity of the absorbance. Atomic force microscopy (AFM) measurements were made in air using Digital Instruments Multimode Nanoscope IV (Bruker AXS) in tapping mode. A silicon cantilever (Nanoworld, NCH-10T; length, 125 lm; tip radius, Rc = 8 nm) was used for imaging. The size of the fiber is an average of more than 20 points of the fibers. The standard deviation of LE and LK were 6.4% and 11.6%, respectively. A literature-based calculation method was used to estimate the plausible size of a flat shaped structure such as a b-sheet peptide fiber while considering the tip convolution effect in AFM imaging [27]. Contact angle measurements were made to determine changes in the hydrophilicity of the mica substrate on their modification by the peptides using an optical contact angle meter (Kyowa Interface Science Co., Ltd., Drop Master 500). A drop of pure water (2 ll) was manually deposited on the peptide-modified surface of the mica substrate. An average contact angle value was obtained by measuring the same sample at five different positions. 3. Results and discussion 3.1. Configuration of peptide adsorbents in aqueous solutions The secondary structure of a peptide containing ionic amino acid residues generally depends on the solution pH. According to the pKa of the amino group [25,26], the side chain of the lysinecontaining peptide is protonated (LK+) at pH 6.0 and deprotonated (LK) at pH 11.0. The CD spectrum of the aqueous solution of LK+ at pH 6.0 showed a negative peak at 195 nm, which is typical for a random-coil structure (Fig. 1A) [28]. When the LK solution was prepared at pH 11.0, the CD spectrum showed a negative peak at 215 nm and a positive peak at 195 nm (Fig. 1A). LK formed a typical b-sheet conformation when the side chain was deprotonated [28]. The pKa values of the carboxylic acid [24,25] show that the side chain of the glutamic acid-containing peptide is protonated (LE) at pH 3.0 and deprotonated (LE ) at pH 12.0. The CD spectrum of the aqueous solution of LE at pH 3.0 showed a negative peak at 215 nm and a positive peak at 195 nm, indicating a typical b-sheet conformation (Fig. 1B). The CD spectrum of LE at pH 12.0 showed a negative peak at 198 nm, which is typical for a random-coil structure (Fig. 1B). Both peptides adopted a highly dispersed configuration as random-coils in aqueous solution when the side chain was charged. 3.2. Adsorption of an amphiphilic peptide containing cationic amino acid residues The 2D self-assembly of LK+, which is designed with a cationic amino acid as a hydrophilic residue, was investigated. A mica substrate was immersed in the solution of LK+ with a random-coil conformation. The AFM image of the mica substrate immersed for 18 h as a typical sample showed fiber-like objects, 23–35-nm width and 5-nm height (Fig. 2A). When a mica substrate was immersed in an aqueous solution of LK at pH 11.0, in which the peptide adopted a b-sheet conformation, 10-nm-wide fibrous objects were observed on the mica surface (Fig. 2B). The width and height of these objects were estimated to be 13.6 and 1.4 nm, respectively. Considering the radius of curvature of the AFM probe, the width was calculated to be 4.6 nm. This value agreed with the expected length of a b-sheet peptide with 16 residues, i.e., 5.6 nm. The height of the fibrous object was twice as high as the monolayer of the peptide with the b-sheet conforma-
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Fig. 1. CD spectra of (A) aqueous solution of (a) LK+ at pH 6.0 and (b) LK at pH 11.0. (B) Aqueous solution of (a) LE at 3.0 and (b) LE at pH 12.0.
Fig. 2. AFM images of adsorbed (A) LK+ at pH 6.0 and (B) LK at pH 11.0 on mica substrate; immersion duration: 18 h; height scale: 10 nm.
tion. Amphiphilic peptides designed with alternating hydrophilic/ hydrophobic amino acids often form bilayered fibrous objects with inward-facing hydrophobic residues. The observed fiber on the mica surface was attributed to the adsorption of the b-sheet peptide fiber formed in the solution. The observed object from LK+ solution was apparently large both in the width and height compared with the b-sheet peptide fiber. It was suggested that the 2D self-assembly according to the adsorption of LK+ was proceeding by complicated manner. The CD spectrum of the adsorbed peptide on the quartz substrates obtained by immersing in the LK+ solution at pH 6.0 showed negative peak around 211 nm (Fig. 3A). In the case of the LK peptide adsorbed quartz substrates, a negative peak was observed at 215 nm, which is typical for a b-sheet conformation. The negative peak obtained from the LK+ solution is slightly shifted to a shorter wavelength from the typical peak of the b-sheet peptide.
Such a peak shift was previously proposed for the mixed randomcoil and b-sheet conformations [28]. The FT-IR spectra of the peptide layers obtained from the LK and LK+ solution were observed (Fig. 3B). The layer obtained from LK solution where the peptide adopted b-sheet conformation showed 1629 cm 1 in the amide I region (solid line in Fig. 3B) [29]. This peak typically assigned to b-sheet conformation supported the LK layer was composed with b-sheet fibers. On the other hand, the LK+ layer showed the peaks at 1645 cm 1 attributed to random-coil (dashed line in Fig. 3B). A small peak at 1626 cm 1 was also observed in this spectrum. This peak corresponded to b-sheet conformation. The observed amide I bands indicated that the planar structure obtained by LK+ adsorption comprised a mixture of b-sheet and random-coil structures. In the case of the combination of oppositely charged cationic adsorbent and anionic substrate, the ionic attractive force dominated the adsorption on the substrate surface. The cationic
Fig. 3. (A) CD spectra of (a) LK+ and (b) LK adsorbed on quartz substrates. (B) FT-IR spectra of (a) LK+ and (b) LK adsorbed on CaF2 substrates.
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residues already exposed to the outer phase in the random-coil configuration readily contact the surface of the substrate and stabilize the adsorbed state as a random-coil. As the ionic interaction progresses, the hydrophobic residues containing eight residues in the 16-mer peptide are inevitably exposed to the outer phase, though the exposure of the hydrophobic residues is energetically unfavorable in aqueous medium. Then, a second layer is formed by the adsorption of additional LK+ molecules from the bulk solution to cover the exposed hydrophobic phase. The constructed large fibrous objects formed by LK+ adsorption can be explained by the complex self-assembly, which involves both the adsorption of the random-coil and the secondary structural transition into the b-sheet structures. In this multidimensional propagation, a secondary structural transition may be induced to form a totally stable planar structure accompanying the topological effect of the peptide self-assembly. It is noteworthy that the obtained LK+ layer on the mica substrate was apparently water-repellent (Fig. 4). The contact angle of the LK+ layer on the mica substrate was 95.2°. This value was much larger than that of the freshly cleaved mica surface (7.0°). An increase in the contact angle was also observed when the immersion duration was short as 1 s. The cationic peptide immediately covered the mica surface and made it water-repellent.
3.3. Adsorption of an amphiphilic peptide containing anionic amino acid residues The 2D self-assembly behavior when LE peptide adopting a random-coil conformation adsorbed on the surface was investigated. The AFM images showed fibrous objects on the surface of the mica substrate (Fig. 5). The width of the fibers was estimated to be 11.9 nm. Considering the radius of curvature of the AFM probe, the estimated width was calculated to be 5.8 nm. This value agreed well with the expected length of a b-sheet peptide with 16 residues, i.e., 5.6 nm. The height of the fibers was 0.6 nm (Fig. 5F), which corresponds to the thickness of a single-layered b-sheet peptide comprising L and E residues adopting an extended state such as a b-sheet conformation. The peptide fibers occupied the surface of the substrate as a function of the immersion time (Fig. 5A–E). This result suggests that the secondary structure of LE dissolved as a random-coil spontaneously transformed into a b-sheet when adsorbed on the mica substrate. The mica substrate was immersed in the peptide solution with pH 3.0; in such a solution, the peptide formed a b-sheet conformation. Fibrous objects were sporadically observed in the AFM images (Fig. 6). Their width and height were estimated to be 14.3 and
Fig. 4. Photograph of the water drops on the LK+ peptide coated mica substrate.
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1.3 nm, respectively. The plausible width value was calculated to be 5.6 nm, which was agreed well with that of the b-sheet fiber comprising 16 residues. The height of the fiber coincided with that of a bilayered object. Like the LK peptide, the LE peptide adopting b-sheet conformation also forms a bilayered fibrous object by facing hydrophobic residues in the solution. The adsorption of the bilayered fiber was very poor compared to the LE adsorption at the same duration of the immersion (Fig. 5E). It suggests that the bilayered structure of the LE peptide with a b-sheet conformation exposing the hydrophilic residues to the outer surface has a low affinity for the hydrophilic mica surface. The CD spectrum of the peptide adsorbed on the quartz substrates immersed in the LE solution at pH 12.0 showed negative and positive peaks at 215 and 195 nm, respectively (Fig. 7A). The adsorbed peptide obtained from the pH 3.0 solution also showed negative and positive peaks at 217 and 195 nm, respectively (Fig. 7A). Both spectra typically indicate a b-sheet conformation. The CD spectra showed that the random-coil/b-sheet transition occurred when the LE peptide adsorbed on the surface of the quartz substrate. The FT-IR spectrum of the peptide layer obtained from the random-coil solution showed peaks at 1542, 1631 and 1698 cm 1 (solid lines in Fig. 7B). These amide I and II bands indicate that the peptide layer adsorbed on the CaF2 substrates is composed of antiparallel b-sheet structures. These bands were also observed for the adsorbed peptide layer obtained from the b-sheet peptide solution at pH 3.0 (Fig. 7B). The results indicate that the peptide in the random-coil state apparently transforms its secondary structure to an antiparallel b-sheet at the solid/water interface. In the FT-IR spectrum obtained from the LE solution, additional peaks were observed at 1578 and 1467 cm 1 (dashed lines in Fig. 7B). The carboxylate salt shows asymmetrical and symmetrical stretching bands around 1570 and 1460 cm 1, respectively [30,31]. The observed peaks, which are peculiar for the peptide layer obtained from the random-coil solution, correspond to the infrared absorption of COO of the glutamic acid side chains. This suggests that b-sheet fibers were formed regardless of anionic repulsion at the interface, and the anionic species derived from the ionized carboxyl side chains were maintained in the 2D self-assembling system. To verify the effect of the ionic species on the 2D self-assembly, an immersion experiment with salt was performed; generally, ionic repulsion is decreased by the addition of salt. A solution of LE with KNO3 (10 equivalents to the anionic residue) was freshly prepared, and the mica substrate was immersed in this solution for 18 h. Nanoscaled fibers were also observed in the AFM images (Fig. 8). Their width and height were in agreement with those of the b-sheet peptide. In the AFM image, the fibrous objects were sporadically layered. The height of the layered region was 1.3 nm, which was equivalent to the height of 2 b-sheet layers. The peptide adsorbed on the substrate first vertically aggregated because of the reduced ionic repulsion, and then transitioned from a random-coil to b-sheet conformation in the first layer of the fibrous object. The orientation of the obtained monolayer of b-sheet fiber was estimated by contact angle measurement. The contact angle of the peptide monolayer obtained from the random-coil solution was estimated to be 15.9°. For comparison, orientation-controlled zpeptide monolayers were prepared conventionally by self-assembly at the air/water interface. When the amphiphilic peptide adopting a b-sheet conformation spreads at the air/water interface, it orients the hydrophobic and hydrophilic residues toward the air and water phases, respectively. The Langmuir–Blodgett (LB) method [32,33] enables the fabrication of the amphiphilic peptide monolayer exposing the hydrophobic residues on a substrate by skimming the peptide monolayer from the water surface. On the other hand, the Langmuir–Schaeffer (LS) method [34,35] enables the fabrication of the amphiphilic peptide monolayer exposing
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Fig. 5. (A–E) AFM images of adsorbed LE at pH 12.0 after 1–18 h; height scale: 5 nm. (F) Section analysis with a line graph of the segment shown in E.
Fig. 6. (A) AFM image of adsorbed LE at pH 3.0 after 18 h; height scale: 5 nm. (B) Section analysis with a line graph of the segment shown in A.
the hydrophilic residues, because the monolayer constructed on the water surface adheres to the horizontally approaching substrate. Both LB and LS films were prepared on mica substrate by spreading LE peptide on the air/water interface. The contact angles of the LB and LS films were estimated to be 42.4° and 16.8°, respectively. The contact angle of the peptide monolayer obtained by the adsorption-based method from the random-coil
solution (15.9°) nearly coincided with that of the LS film. Accordingly, it is conceivable that the orientation of the peptide monolayer constructed by the adsorption of the random-coil was controlled with the hydrophilic residues exposed at the outer surface. Conversely, it indicated that the adsorption of the b-sheet nanofiber was stabilized by the hydrophobic interactions. Stabilization of the random-coil in aqueous solution is achieved
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Fig. 7. (A) CD spectra of (a) LE and (b) LE adsorbed on quartz substrates. (B) FT-IR spectra of (a) LE and (b) LE adsorbed on CaF2 substrates.
Fig. 8. (A) AFM image of adsorbed LE at pH 12.0 with KNO3; height scale: 5 nm. (B) Section analysis with a line graph of the segment shown in A.
Fig. 9. Plausible images of the configuration constructed by the amphiphilic peptide (A) in the aqueous solution adopting a random-coil conformation and (B) on the substrate adopting a b-sheet conformation.
by a configuration that hides the hydrophobic residues within the hydrophilic moieties. However, in the random-coil configuration, the hydrophobic residues that occupy half of the amino-acid residues in the 16-mer peptide are sterically crowded, and hence,
partially exposed to the outer surface. Thus, the random-coil dissolved in the aqueous solution inevitably possesses regions with high surface energy. Owing to the nonhomogeneous surface character of the random-coil, the peptide can adsorb onto the
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substrate by hydrophobic interaction. The peptide then transformed to a b-sheet conformation, when the 2D density of the peptide increased over a critical point as the adsorption proceeded (Fig. 9). In the aqueous solution of LE at pH 3.0, the critical aggregation concentration (CAC) was estimated to be 6.8 10–7 M by fluorescence measurements using pyrene. CD measurements showed that the peptide adopted a b-sheet conformation above the CAC. In contrast, the CAC of LE at pH 12.0 was not detected in the aqueous medium. The peptide showed CD spectra supporting only a random-coil conformation in the soluble concentration regime. The surface of the substrate may offer a self-assembling field where the LE can aggregate above a critical value. Then, the adsorbed LE underwent the secondary structural transition to gain stability by inter-strand hydrogen bonding. Such 2D fibrillization based on the b-sheets of the anionic peptide amphiphile occurred in spite of the ionic repulsion between the anionic moieties on the side chain. Such lateral aggregation at the interface probably occurs upon modulating the competition between the stabilizing effects of the interstrand hydrogen bonding and hydrophobic interactions and the destabilizing effect of the ionic repulsion. When the sequential peptide took an anti-parallel b-sheet conformation, the distances between the neighboring side chains in the hydrophilic face (lch) and peptide strands (lstr) were 0.7 and 0.47 nm, respectively [9,36,37]. The value of lch is comparable to value of the Bjerrum length (LB) [38], at which the potential electric energy of two elementary charges is equal to the thermal energy (kBT). The fact that lstr is shorter than LB suggests that the aggregation of the peptide strands is influenced by the ionic repulsion. For instance, when amphiphiles such as fatty acids and lipids form a lamellar structure or monolayer, the distance between the hydrophilic head groups in the 2D close-packed structure is reported to be 0.45–0.49 nm [39,40]. This means that amphiphiles can aggregate within the LB regardless of the ionic repulsion between the hydrophilic heads in aqueous solution, if there are supplemental stabilizing factors such as hydrophobic interactions. Considering that the distance between the peptide strands adopting a b-sheet conformation is approximately equal to the size of the head groups of the amphiphiles, the dimensions of the aggregated peptide with anionic side chains are reasonable. In the 2D environment, the LE formed a b-sheet conformation supplemented by hydrophobic interactions and inter-strand hydrogen bonding, while it took a random-coil conformation in a 3D environment such as a bulk solution, where it was influenced by hydrophobic interactions and ionic repulsion. 4. Conclusions We have demonstrated the spontaneous construction of a 2D layered architecture by the adsorption of specially designed amphiphilic peptides with ionic residues as hydrophilic moieties. A sequential peptide comprising cationic hydrophilic/hydrophobic amino-acid residues formed a water-repellent planar architecture. The obtained architecture was based on a mixture of random-coil and b-sheet conformations. These results show that the combination of oppositely charged peptide and substrate stabilized a relatively rigid topological structure based on ionic attractive forces. On the other hand, a sequential peptide designed with a series of anionic hydrophilic/hydrophobic amino acid residues underwent a random-coil/b-sheet transition at the substrate/water interface, yielding a nanoscale fiber array. The obtained structure was an orientation-controlled monolayer, in which the hydrophilic residues are exposed at the outer surface. Interestingly, even though both the peptide and substrate are anionic, the adsorption of the peptide actually resulted in a monolayered b-sheet fiber array. This
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