Peptides as Smart Biomolecular Tools: Utilization of Their Molecular

Chapter 3

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Peptides as Smart Biomolecular Tools: Utilization of Their Molecular Recognition for Materials Engineering Toshiki Sawada and Takeshi Serizawa* Department of Chemical Science and Engineering, School of Materials and Chemical Technology, Tokyo Institute of Technology, 2-12-1-H121 Ookayama, Meguro-ku, Tokyo 152-8550, Japan *E-mail: [email protected]

Biomolecules express outstanding properties required for molecular recognition and nanoscale self-assembly. These smart capabilities have been obtained through evolution, and these biomolecules utilized in nature based on a smart function. Recently, their excellent capabilities have been utilized in not only biosystems, but also in materials science and engineering for functional materials. The bioinspired peptide selection technology using phage display systems has been developed based on this natural evolution to generate novel functional peptides. In this chapter, we focused on peptides with a specific affinity for synthetic polymers. The polymer-binding peptides were obtained from a biologically constructed phage-displayed peptide library to recognize polymeric nanostructures, and were utilized for possible applications as novel biomolecular tools for functional polymers. Our approach to utilize peptides as novel biomolecular tools will open excellent opportunities for the next-generation of materials science and engineering.

1. Introduction Natural biomolecules such as peptides, proteins, nucleic acids, and saccharides exhibit the properties required for molecular recognition and nanoscale self-assembly. Such biomolecules have been constructed through evolutional systems, and functioned based on smart molecular recognition. © 2017 American Chemical Society Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Molecular recognition plays an important role in natural systems, and can be observed between antigen-antibody (1, 2), DNA-protein (3, 4), RNA-ribosome (5), and sugar-lectin (6) interactions to sustain biosystems. These molecules have undergone structural-fitting through evolution to appropriately combine numerous weak interactions (noncovalent bonds) such as electrostatic, hydrogen bonding, π-stacking, van der Waals interactions, and hydrophobic effects. The targets of these biomolecules have been considered to naturally occurring ligands present in the biological systems. However, recent studies have revealed certain peptides with specific affinities for artificial materials through bioinspired affinity-based selection procedures from biologically constructed peptide libraries displayed on phages or cell surfaces (7–10). The surfaces of metals, metal oxides, semiconductors, magnets, nanocarbons, synthetic polymers, and artificially designed peptide assemblies were utilized as the specific targets of these novel peptides. These peptides have a regular structure, and therefore might recognize two- or three-dimensional regular distributions of atoms or functional groups on the material surfaces, thus resulting in specific affinities. These studies suggested that biomolecules, particularly peptides, could be evolved toward specific interactions for artificial materials by suitable selection procedures that mimics the natural evolutionary system. Recently, due to limitations in cell surface display systems for the selection against artificial materials, phage display systems have been widely utilized for construction of material-binding peptides. These peptides are known not only as material binders, but also as functional molecular tools for a wide range of applications such as surface modifiers, adsorbents for patterning, catalysts for the preparation of inorganic particles, constructs of polymeric nanoparticles, and switchers for conjugated polymer fluorescence. Since the affinities of the peptides were effectively utilized to achieve all of the above applications, the molecular recognition capabilities of these peptides will be effective for constructing novel classes of peptide-based nanomaterials.

Figure 1. Possible polymeric nanostructures for peptide recognition. Reprinted with permission from ref (11). Copyright 2011 Royal Society of Chemistry. 32 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


The purpose of this chapter is to overview the advances that have been made using specific interactions through molecular recognition for artificial materials (especially synthetic polymers) for fabricating a novel class of biomolecular tools by our group over the past decade. Indeed, we have screened, characterized, and utilized polymer-binding peptides against polymeric materials using phage displayed peptide libraries (11, 12). We are forcusing on peptides that discriminate against slight structural differences in synthetic polymers. A primary sequence, stereoregularity, amphiphilicity, crystallinity, porosity, a linear/branched structure, and an assembled structure may fit into the three-dimensionally regular nanostructures of certain peptides (Figure 1). Nanomaterials composed of peptides using those excellent capabilities will open attractive opportunities for the science and technology of next-generation biomolecular tools and nanomaterials.

2. Selection of Polymer-Binding Peptides 2.1. Synthetic Polymers as Peptide Targets It is crucial to understand and regulate biological phenomena such as adsorption of proteins and adhesion of cells on the surface of synthetic polymers at the molecular level for developing the novel polymer materials utilized in biomedical fields (13–17). In the case of hydrophobic or charged polymers, proteins can easily adsorb onto polymer surfaces, thereby mediating various biological processes such as cell adhesion, extension, and proliferation. These interfacial phenomena is normally hard to understand, due to multiple and complicated weak interactions at the interfaces. Hence, we originally focused on more reliable interactions of biomolecules against the polymer surfaces. Furthermore, it is also interesting whether biomolecular peptides can recognize nanostructures derived from the characteristic of synthetic polymers, which are typically stereoregularity, amphiphilicity, crystallinity, and so on. In the area of inorganic materials, Belcher and co-workers described peptides discriminating crystal defects in germanium thin films on silicon and germanium wafers, suggesting that the recognition capability of peptides could be utilized for nondestructively probing and identifying the localization of defects in crystalline substrates (18). The result indicated that the molecular recognition capability of artificially evolved peptides could be exploited in materials engineering. Although applications of material-binding peptides have been limited at solid-liquid interfaces due to their selection conditions, peptides with specific affinities for water-soluble polymers would show novel potential utilization in solutions. Thus, peptides that can discriminate slight differences in polymeric nanostructures will open attractive new opportunities for polymer science and technology. In 1985, it was reported that desired peptides could be genetically displayed on the coat proteins of filamentous phages based on the insertion of the corresponding DNA fragment into the phage genome (19). The infectiousness of the resultant genetically engineered phages against a host Escherichia coli (E. coli) was maintained. Thus, the genetically engineered phages could be easily 33 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


amplified. One of the most commonly utilized phages is M13 filamentous phage, which is composed of five kinds of coat proteins termed pIII, pVI, pII, pVIII, and pIX on the surface (Figure 2). When peptides with different amino acid sequences are displayed on the surface of each different phage, a phage-displayed peptide library can be constructed in principle. Recently, the phage display method is a versatile and important tool for the selection of ligands for biomolecules such as peptides and proteins (20, 21). This approach has also been accepted over the past decade to select material-binding peptides. The selection and characterization of such phage-displayed material-binding peptides has attracted great interest, especially due to their wide utilization in material engineering and nanotechnology. The protocols for the construction of phage-displayed peptide libraries have been established (21, 22). The bioinspired selection process using a peptide library displayed on phages to target-specific phage pools is called biopanning (Figure 3). Biopanning is composed of four fundamental experimental steps as described below. Briefly, step 1 is the interaction in which an aqueous solution of the phage libraries is mounted onto the target material surfaces (solid such as film, particle, and so on) for an adequate time. In step 2, the washing, the target material surfaces are washed with a buffer solution (sometimes containing detergents) several times to remove unbound or weakly bound phages. In step 3, the elution, the strongly bound phages are eluted from the material surfaces by an eluent (acid solutions are usually used). In step 4, the amplification, the eluted phages are then proliferated within E. coli for amplification. After the appropriate number of biopanning cycles, cloning of the selected phages followed by the DNA sequencing of the displayed peptides on each phage clone, which corresponds to each peptide sequence, is performed.

Figure 2. Schematic illustration of M13 filamentous phage displaying peptides on pIII. Reprinted with permission from ref (12). Copyright 2013 Wiley. 34 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 3. Schematic illustration of the screening process using a phage-displayed peptide library. Reprinted with permission from ref (12). Copyright 2013 Wiley. 2.2. Recognition of Polymer Stereoregularities by Selected Peptides 2.2.1. Selection and Characterization of Stereoregular Polymer-Binding Peptides For the first target, peptide selection using a phage library with 7-mer random peptides was performed against a stereoregular polymer, isotactic poly(methyl methacrylate) (it-PMMA) (Figure 4a), in which the side chains are almost all on one side of the backbone (Figure 5a) (23). In the case of this target polymer, syndiotactic (st-) PMMA, in which the side chains on alternate sides, was used as a reference polymer. After five rounds of biopanning for it-PMMA spin-cast film, nine phage clones were identified. Enzyme-linked immunosorbent assay (ELISA) using the phage clones revealed that the binding amounts of all phage clones for itPMMA were much greater than those for st-PMMA. The ELISA result suggested that the four amino acid peptide motif (Arg-Pro-Thr-Arg) composed of amino acids with proton-donor hydroxyl and amino lateral groups adjacent to the Pro was an essential for both affinity and specificity. Considering the high molecular weight of the M13 phage (16 300 000) (24), it was surprising that the 7-mer peptides displayed on the filamentous phage termini greatly affected the binding capability of the phage clones. 35 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 4. Chemical structures of synthetic polymers applied to the peptide screening. (a) Poly(methyl methacrylate) (PMMA), (b) poly(L-lactide) (PLLA), (c) polystyrene(PS), (d) linear (top) and branched (bottom) poly(p-phenlene vinylene) (PPV), (e) Polyetherimide (PEI), (f) poly(propylene oxide) (PPO), (g) poly(2-methoxy-5-propyloxysulfonate-1,4-phyenylenvinylene), and (h) poly(N-isopropylacrylamide) (PNIPAM).

Figure 5. (a) Recognition of stereoregular it-PMMA films by the c02 peptide with a sequence of Glu-Leu-Trp-Arg-Pro-Thr-Arg. The C-terminal 4-mer peptide (Arg-Pro-Thr-Arg, RPTR) was essential for the specific binding. (b) Comparison of the possible structures of RPTR obtained by Molecular Mechanics and hexa MMA units. Reprinted with permission ref (23). Copyright 2005 ACS and ref (25). Copyright 2007 ACS.

36 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


The binding of the chemically-synthesized 7-mer peptides for the target itPMMA and the reference st-PMMA were monitored in a real time and kinetically analyzed by surface plasmon resonance (SPR) measurements, and their kinetic parameters were determined (25). The binding constant (Ka) values of the c02 peptide with the sequence Glu-Leu-Trp-Arg-Pro-Thr-Arg approached 2.8 × 105 M-1 for the target, which was 40 times greater than that for the reference polymer (6.9 × 103 M-1). The value for the target was comparable to the Ka values of 12-mer peptides that bind to titanium oxide surfaces (26), suggesting that peptides have the potential to discriminate subtle differences in the stereoregularity of polymeric structures. Detailed analyses by Ala-scanning (binding analyses using substituted peptides, in which each amino acid was changed to Ala) clearly demonstrated that the Ka values of the Ala-substituted peptides decreased significantly. The smallest Ka value was more than 30 times less than that of the original c02 peptide. The magnitude of the decrease of the Ka values was as follows: Pro > Thr > Arg7 > Glu > Arg4, suggesting that these amino acids were essential for the specific binding. These observations from the Ala substitution directly evidenced the importance of the recognition of the polymeric structures by amino acid side chains. A rigid conformation of the peptide derived from a kinked Pro residue should provide three-dimensionally arrange the lateral hydroxyl and amino groups of the Thr and the two Arg residues as proton-donors to successfully recognize the position of the it-PMMA ester groups through hydrogen bonding. To check the importance of the it-PMMA binding motif, the Ka values of the N-terminal (Glu-Leu-Trp-Arg) and C-terminal (Arg-Pro-Thr-Arg) peptides for itand st-PMMA were determined. The Ka of the C-terminal peptide for it-PMMA (4.6 × 104 M-1) was greater than that for st-PMMA (9.4 102 M-1) and that of the N-terminal peptide for it-PMMA (1.6 × 103 M-1). Therefore, it was demonstrated that the C-terminal 4-mer sequence of the c02 peptide, Arg-Pro-Thr-Arg, which is composed of proton-donor hydroxyl and amino lateral groups adjacent to the Pro, was an essential motif for it-PMMA recognition, and that peptides composed of four amino acid residues have the enough potential to recognize polymer stereoregularity (Figure 5a). Possible structures with stable and sterically acceptable conformations of the 4-mer motif were obtained by energy optimization using Molecular Mechanics, and the obtained size of the structural conformation was comparable to the size for 6 units of MMA (Figure 5b). To further characterize these peptide recognition mechanisms, thermodynamic parameters for the c02 peptide against it- and st-PMMA films were determined by analyzing the Ka value at various temperatures (25). The Ka value decreased with increasing temperature, suggesting that the predominant interactions of the peptide affinities for it-PMMA films was derived from hydrogen bonding interactions. The estimated enthalpy (ΔH°) and entropy (ΔS°) changes assuming the van’t Hoff equation suggested that greater conformational changes of the c02 peptide and/or it-PMMA occurred to obtain the specific affinity. In other words, the specific affinities seemd to be derived from an induced fit mechanism. Considering all of the results, it was clearly demonstrated that short peptides had the potential capability to discriminate polymeric nanostructures through specific binding as natural biomolecules did (Figure 5). 37 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


2.2.2. Generality of Peptide Recognition for Stereoregular Polymers

Previously utilized as a reference polymer (st-PMMA) was applied to the next target of the selection using a phage-displayed linear 7-mer peptide library to determine whether peptides can generally recognize polymeric nanostructures derived from polymer stereoregularity (27). Furthermore, because the surface accumulation of functional groups on st-PMMA films are affected by external environment, st-PMMA is an attractive interesting as a novel peptide target. It is well known that when st-PMMA films are prepared on glass slides in air, hydrophilic ester and hydrophobic alkyl groups are accumulated on the glass and air sides, respectively (28). Therefore, st-PMMA films with variable surface properties are suitable candidates for demonstrating both the potential specificity of peptides against materials and the novel strategy to control interactions between the peptides and softmaterials. Therefore, st-PMMA films were prepared on glass surfaces and immersed in a buffer for 15 h (conditioning). This treatment resulted in the exposure of the ester groups to the air side. After three rounds of biopanning for the conditioned st-PMMA films, several phage clones were identified. DNA sequencing revealed the phage clones displaying peptides composed of several amino acid residues with amino groups and a Pro residue. Therefore, it was anticipated that hydrogen bonding interactions between the peptides and the st-PMMA were essential for the affinity. ELISA result indicated that one phage clone displaying the peptide with a sequence of His-Lys-Pro-Asp-Ala-Asn-Arg had a specific affinity for the target st-PMMA rather than for the reference it-PMMA. The Ka values of chemically-synthesized peptides for st- and it-PMMA films were determined to 9.1 × 104 M-1 and 3.0 × 103 M-1, respectively. Therefore, it was demonstrated that peptides could generally discriminate subtle differences in nanostructures derived from polymer stereoregularities. More interestingly, the binding amounts of the phage clones for the buffer conditioned st-PMMA films as peptide targets were much greater than those for non-conditioned st-PMMA films. In fact, the static contact angles of the st-PMMA films decreased with increasing conditioning time, suggesting that the hydrophilic ester groups of st-PMMA rather than the hydrophobic alkyl and/or methyl groups were gradually exposed into the water side. Therefore, it was confirmed that peptides can recognize nanoscaled arrangements of ester groups due to the syndiotacticities of the methacrylates. As a consequence, peptide motifs that could recognize variability in polymer film surfaces were discovered based on their selection from peptide-displaying phage libraries directed against adequately-treated st-PMMA films. Furthermore, it is well known that it- and st-PMMAs self-assemble into triple-stranded helix structures, stereocomplexes (SCs), in which the it-PMMA double helices are surrounded by a single st-PMMA helix (29), and the SCs could be prepared on substrates through a stepwise layer-by-layer (LbL) assembly (30). Such structurally defined stereocomplex structures were applied to the selection process using a 7-mer peptide displaying phage library (31). The obtained peptide with a sequence of Ser-Thr-Pro-Pro-Arg-Leu-Trp bound specifically to the SC films as compared to single it- or st-PMMA spin-cast films. Therefore, 38 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

nanostructures prepared from stereoregular PMMAs would generally be definite targets for peptides.


2.3. Recognition of Other Polymer Structures by Peptides Poly(L-lactide) (PLLA) (Figure 4b) is one of the most commonly-used synthetic polymers in biomedical fields due to high thermal stability, mechanical properties, biocompatibility, and biodegradability for non-toxic products (32, 33). However, it is difficult to introduce functionalities onto the surface of PLLA because of its few active groups, thus resulting in limiation of its biomedical applications. Here, α-formed crystalline PLLA was applied to the selection using the 7-mer phage-displayed peptide library toward surface functionalization of PLLA (34). The Ka value of the chemically-synthesized peptide with a sequence of Glu-Leu-Met-His-Asp-Tyr-Arg, which was selected from the library, for the PLLA films determined by SPR kinetic measurements (6.1 × 104 M-1) was 10 times higher than for amorphous PLLA films. Therefore, the obtained peptide clearly recognizes the polymeric structures derived from a simple annealing process that enhances polymer crystallinity. Furthermore, the Ka value for the 103 helical α-form was 4 times higher than that for the 31 helical β-form, indicating that the peptide could discriminate slight differences in the helical pitch delivered from the PLLA morphs. Polystyrene (PS) (Figure 4c) is a universal vinyl polymer frequently utilized as plastic plates for biological experiments. Previously, PS binding peptides were inadvertently obtained (35), even though PS was consisting of simple alkyl main chains and lateral phenyl groups. We focused on specific nanostructures formed by syndiotactic PS (sPS), and applied to the selection procedures using a 7-mer peptide library for the sPS films (36). When the films were prepared from sPS solutions dissolved in a suitable solvent, sPS forms TTGG helical conformations (T and G represent trans and gauche conformations, respectively) (37–39). Evaporation of the free solvent resulted in δ-form films complexed with solvent molecules. Further evaporation under thermal heating conditions fabricated empty δ-form (δe) porous films, and the δe formed sPS films were utilized as peptide targets. The sequence of the selected peptides displayed on the phage clone was Phe-Ser-Trp-Glu-Ala-Phe-Ala composed of aromatic and aliphatic amino acids, suggesting that π-stacking and hydrophobic effects are essential for the sPS binding. In fact, binding amounts of the phage clones determined by ELISA against films composed of target sPS, atactic PS, isotactic PS, and sPS/toluene complexes (δ-form) clearly demonstrated the highly specific affinity of the peptide for the δe form, indicating that peptides have the potential to recognize porous nanostructures composed of synthetic polymers. Conjugated polymers are attractive materials due to their unique optical and electronic properties. Thus, the polymers have been utilized in a number of applications including light emitting diodes (40), photovoltaic cells (41), and biosensing (42). A representative conjugated polymer, poly(phenylene vinylene) 39 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


(PPV) (Figure 4d), which has a simple chemical structure composed of vinyl groups and phenyl rings, is the most extensively studied conjugated polymer. We focused on peptide recognition for linear and branched polymer structures of PPV, and both PPVs were applied to the peptide selection using a phage library with 12-mer peptides (43). The obtained peptides for each target showed specific affinities, indicating that the peptides recognized the structural differences in the PPV isomers. Because aromatic amino acids were observed in sequences as His-Thr-Asp-Trp-Arg-Leu-Gly-Thr-Trp-His-His-Ser, π-stacking interactions between the peptides and the PPV might be essential. Possible molecular structures determined by molecular mechanics suggested that two Trp residues in the peptide gave suitable interactions with the phenyl ring in the branched PPV. Polyetherimide (PEI) (Figure 4e) is an engineering plastic with excellent physicochemical properties, and has been widely utilized in advanced industries. PEI is a candidate for replacement of metallic materials, and thus has been increasingly employed as a biomaterial (44). Since regulating these biological responses as well as adding another function at the PEI surface is essential for the development of softmaterials, PEI binding peptides were selected from a 7-mer peptide library on phages (45). The Ka value of the chemically synthesized peptide with a sequence of Thr-Gly-Ala-Asp-Leu-Asn-Thr for the target PEI films (5.6 × 108 M-1) was much greater than that for non-target thermally treated PEI films (4.4 × 104 M-1). The aforementioned difference in the Ka values must be attributed to the structural differences in the PEI surfaces before and after thermal treatment. Since thermal treatment of the PEI films strongly affected molecular aggregations or stacking of the monomer units, the identified peptides should discriminate the structural changes of the PEI films. Poly(propylene oxide) (PPO) (Figure 4f) is a hydrophobic component for tribrock copolymer composed of a central PPO chain flanked by two hydrophilic chains of poly(ethylene ooxide) (PEO). The triblock copolymers have been widly utilized as emulsifiers and carrier matrices for a drug delivery system (DDS) because they form micelles, followed by physically closs-linked hydrogels depending on their concentrations. Because it is difficult to functionalize the triblock copolymer due to their few active groups, further applications in biomedical fields are limited. Therefore, we selected peptides with specific affinity for PPO using the 12-mer peptide library (46). After 2 rounds of biopanning, single phage clone displaying peptides with a sequence of Asp-Phe-Asn-Pro-Tyr-Leu-Gly-Val-Thr-Pro-Val-Lys. The Ka value of the peptide for PPO films (1.2 × 106 M-1) is much higher than that for reference PMMA films (3.6 × 104 M-1), indicating specific affinity for PPO. Time dependent release of the peptides from the hydrogels composed of PEO-PPO-PEO was investigated, and the release properties were correlated with the affinities of the peptide, indicating certain binding of the peptide for the target segment PPO even in the hydrogels. Because the aforementioned synthetic polymers used as peptide targets are water-insoluble, the applications for these polymer-binding peptides would be limited to solid surfaces. Therefore, we applied to the peptide selection method to films composed of water-soluble poly(2-methoxy-5-propyloxysulfonate-1,4phenylen vinylen) (mpsPPV) (Figure 4g) using a layer-by-layer (LbL) assembly technique with poly(diallyldimethylammonium chloride) (PDDA). The identified 40 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


peptide with a sequence of His-Asn-Ala-Tyr-Trp-His-Trp-Pro-Pro-Ser-Met-Thr bound to not only the LbL films, but also to dissolved mpsPPV in aqueous solution (47). These results indicated that a wide variety of water-soluble polymers could be peptide targets. Poly(N-isopropylacrylamide) (PNIPAM) (Figure 4h) is a representative classic thermoresponsive water-soluble polymer and show a reversible coil-grobule transition at the lower critical solution temperature (LCST). The LCST of PNIPAM is near the body temperature; therefore, PNIPAM has been widely used in biomedical fields such as scaffolds to construct cell sheets and thermorespovsive precipitation of desired proteins. Because the interaction of biomolecular ligands into PNIPMA chains through chemical modification is sometimes complicated and time consuming, we selected 12-mer peptides with a specific affinity for water insoluble PNIPAM with high meso diad content (85%) from the peptide library (48). The identified chemically synthesized peptides showed higher Ka value for the PNIPAM films (2.0 × 105 M-1) rather than for it-PMMA films (1.7 × 104 M-1), indicating specific affinity for the PNIPAM. Furthermore, the peptide specifically lowered the LCST of meso-rich (58%) PNIPAM rather than meso-poor PNIPAM, demonstrating the peptide preferentially bound to the mesosequence of PNIPAM chains even in an aqueous phase. As mensioned above, we have successfully screened peptides with spefic affinities for various synthetic polymers (Table 1). The peptides clearly discriminated unique polymeric structures such as stereoregularity, amphiphilic structures, crystallinity, porous structures, linear/branched structures, and so on. Because such artificial nanostructures derived from synthetic polymers were recognized by biomolecular peptides, further various polymers would be applied to the screening procedure to obtain their specific peptides for future applications.

3. Applications of Polymer-Binding Peptides Functionalizaon of material surfaces plays an essential role in material science and engineering. In various surface modification methodorogies, it is usually used to form self-assembled monolayers (SAMs) on inorganic material surfaces for surface modification, both practically and experimentally, because of the simpleness for preparation of functional surfaces with well-defined compositions on substrates (49–51). If other softmaterials could be functionalized by such a concept of SAMs, the range of technical applications would be broaden. Therefore, we anticipitated peptides with specific affinities for the synthetic polymers were used for surface modification towards biomedical applications (Figure 6a). As target substrates, PEI (45) and PMMA SC (31) films were utilized for surface modification through their specific peptide. Biotin with extermely high affinity for streptavidin (SAv) was used as a functional moiety conjugated to the specific peptides. For the immobilization of SAv, the biotin-conjugated 41 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


peptides were immobilized onto the polymer surfaces. The amount of bound SAv was dependent on the density of the immobilized biotin-conjugated peptides, suggesting that SAv molecules were immobilized onto the substrate through the biotin molecule conjugated to the peptides. The immobilized SAv through the biotin-conjugated peptides could be utilized to further immobilization of probe DNA to efficiently hybridize with its complementary DNA as compared to directly immobilized SAv on the PEI films. The SAv immobilized through the biotinylated specific peptide formed uniform monolayers without denaturing on the PEI films. Therefore, it was demonstarated that polymer-binding peptides had great potential for the functional modification of polymer surfaces.

Table 1. Summary of characterization of polymer-binding peptides Polymer

Ka/ 105 M-1

Sequence

Specificity

Target

Reference

it-PMMA

ELWRPTR

2.8

0.069 (st-PMMA)

41

st-PMMA

HKPDANR

0.91

0.030 (it-PMMA)

30

PLLA (α-form)

ELMHDYR

0.61

0.057 (Amorphous PLLA)

11

Linear PPV

ELWSIDTSAHRK

0.77

0.36 (Branched PPV)

2.1

Branched PPV

HTDWRLGTWHHS

7.7

0.52 (Liner PPV)

15

mpsPPV

HNAYWHWPPSMT

1.3

-

-

PEI

TGADLNT

5600

0.44 (Thermally treated PEI)

13000

PPO

DFNPYLGVTPVK

12

0.36 (PMMA)

33

PNIPAM

HSFKWLDSPRLR

2.0

0.17 (PMMA)

12



Figure 6. (a) Representative applications of polymer-binding peptides: (a) polymer surface modification by proteins through the peptides, (b) polymer-binding peptide-fused proteins, (c) peptide capped gold nanoparticles, and (d) conjugated polymer nanoparticles hybridized to the polymer-binding peptides with and without staining of peptides. Scale bars in (c) and (d) represent 100 nm. (e) Alive and dead cells incubated with the Dox-conjugated peptides or the original peptides released from polymeric hydrogels. Scale bars represent 100 µm. (f) Thermoresponsive precipitation of the peptide-fused HSA in the presence of thermoresponsive polymers. Reprinted with permission from (c) ref (63). Copyright 2009 ACS, (d) ref (70). Copyright 2011 Royal Society of Chemistry, (e) ref (46). Copyright 2016 Royal Society of Chemistry. In the previous paragraph, a functional protein (SAv) was immobilized through the biotin-conjugated polymer-binding peptide. Another protein immobilizaition strategy on surfaces is to fuse the peptide for the functionalization of protein directly. In fact, apoferritin (52, 53), cytokines (54), and green fluorescent protein (55) have been fused with material binding peptides for immobilization through specific interactions. These proteins have the capability to be stably immobilized on inorganic and nanocarbon materials through noncovalent but specific interactions. We also prepared proteins fused with the polymer binding peptides for enhanced adsorption against polymeric substrates. The c02 peptide, which binds specifically to the it-PMMA films (Figure 6b) (56), was conjugated to DnaK419-607, a blocking peptide fragment (BPF) which is part of the substrate binding domain of the molecular chaperon DnaK from E. coli (57, 58), to construct an effective blocking reagent due to its excellent adsorptive 43 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


properties for plastic substrates through its hydrophobic domain (59). The c02 peptide was fused to the N-terminus of the BPF derivative (dBPF). The Ka of the c02 peptide-fused-dBPF for it-PMMA was 4.9 × 108 M-1, which was 75 times greater than that of the original dBPF. Thus, the fusion protein showed specific affinity for it-PMMA, and the specificity was due to the fused c02 peptide. Furthermore, other functional molecules were also specifically immobilized onto other kinds of substrates. Gold nanoparticles (GNPs) have potential applications in the biological and nanotechnological fields due to their unique surface plasmon properties (60–62). Therefore, the surface functionalization of GNPs is required to widen their applicabilities against various fields. We prepared GNPs with a specific affinity for it-PMMA by capping of peptide with the sequence of Arg-Pro-Thr-Arg, which is an essential motif for the it-PMMA affinity (25). Cys was fused to the N-terminus of the motif as an anchor for the gold surface through strong thiol-Au binding. AuCl4- was reduced in a 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer containing the designed 5-mer peptide with a sequence of Cys-Arg-Pro-Thr-Arg, under mild conditions (pH 7.2, ambient temperature), resulting in peptide-capped GNPs, which were stably dispersed in HEPES buffer solutions (Figure 6c) (63). The prepared nanoparticles showed preferentialy affinity for it-PMMA film surface as compared to st-PMMA surfaces, insicating that polymer-binding peptides could be used as anchor molecules for the heterogeneous interface between inorganic compounds and synthetic polymers. Peptide-capped nanoparticles with cores of gold (64, 65), silver (66), platinum (64), silica (67), and polymeric nanogels (68) have been prepared and studied for biomedical and bio-analytical applications. Moreover, conjugated polymer nanoparticles (CPNs) are gaining attention because of their excellent optical and electronic properties (69). Although the desired surface functionalization of CPNs would expand potential capabilities utilized in optical and biomedical fields, only covalent functionalization methods have been reported thus far. Therefore, we have tried to construct peptide-capped CPNs through noncovalent specific interactions. CPNs composed of water-insoluble, branched PPV and its specific peptide were prepared by simply mixing of the PPV dissolved in an organic solvent into an aqueous solution of the specific peptide, and subsequent sonication processes (Figure 6d) (70). Since the peptides were internalized into the CPNs, the CPNs were clearly different from conventionally prepared polymer nanoparticles. This is the first demonstration of utilization of a specific binding peptide for the preparation of polymeric nanoparticles. To demonstrate the biomedical applicability of the polymer-binding peptide, the PPO-binding peptide was modified with an anticancer drug molecule, doxorubicine (Dox), thorugh suitable linker molecules to construct a controlled release system of drugs from the PEO-PPO-PEO tribrock copolymer hydrogels. The release rate of the Dox-conjugated peptides was slightly smaller than that of the original PPO-binding peptide under the same conditions, indicating that the specific affinity was remained after modification with hydrophobic Dox. The release system was applied to cell culture assays to clarify effectiveness of the controlled release system. Numbers of alive HeLa cells were successfully decreased with increasing incubation time in the presence of the Dox-conjugated 44 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


peptide-containing PEO-PPO-PEO hydrogels, demonstrating sustainable anticancer effects (Figure 6e) (46). Therefore, it was demonstrated that controlled release systems using polymeric hydrogels and specific peptides could be utilized for future biomedical applicaations. The peptide affinity for water-soluble thermoresponsive polymers was utilized to precipitate desired proteins. Human serum albumin, a model protein, was chemically modified with the PNIPAM-binding peptides through a disulfide bond to specifically interact with the meso-rich PNIPAM dissolved in water. The Ka value of the peptide-modified human serum albumin (HSA) for the target PNIPAM was 4.4 × 107 M-1, which was 17 times greater than that of native HSA, indicating that peptide on HSA molecules still showed an affinity for the meso diad sequence of PNIPAM. Thermoresponsive precipitation experiments using meso-rich PNIPAM and HSA modified with or without peptides were performed. As a result, 98% of the peptide-modified HSA was precipitated with the meso-rich PNIPAM, even though approximately 20 % of unmodified HSA was precipitated with the meso-rich PNIPAM (Figure 6f) (48). Hence, the PNIPAM-binding peptide-modification was useful to thermoresponsively precipitate and collect desired biomacromolecules with PNIPAM.

4. Conclusion Recent developments in synthetic polymer-binding peptides that can recognize the nanostructures of synthetic polymers were described. Our observations demonstrated that polymeric nanostructures derived from unique stereoregularity, amphiphilicity, crystallinity, porosity, linear/branching structure, and dynamic comformational changes could be targets for the peptide. The use of these polymer-binding peptides for surface functionalization through noncovalent peptide or fusion protein interaction, metal nanoparticle functionalization with novel synthetic systems, a one-pot synthesis of polymer-peptide conjugated particles, controlled release systems from polymeric hydrogels, and thermoresponsive precipitation of proteins were achieved. In all cases, peptide recognition against the synthetic polymers was effectively utilized. Therefore, it was evidenced that biomolecular peptides could be versatile molecular tools for the development of novel material innovations. Biomolecular peptides have great potential for use in the materials science and engineering field, and are superior to native biological functions. Bioinspired selection technology using phage display systems has exploited the natural process of material evolution to create a new generation of bionanomaterials with novel functions. These excellent characteristics of peptides and strategies for the construction of bioinspired peptidyl molecular tools will open attractive opportunities for the science and engineering of next-generation bionanomaterials.

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Peptides as Smart Biomolecular Tools: Utilization of Their Molecular

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