Hisao Matsuno

Isolation of Peptides that Can Recognize Syndiotactic Polystyrene

Recent progresses in combinatorial biotechnologies such as phage display (PD) and cell-surface display methods have verified the presence of certain short peptides that specifically recognize and bind to artificial material surfaces that are not the original biomolecular targets. The biological selection of clone phages, or cells displaying target-specific peptides from phage or cell libraries (called biopanning), and subsequent DNA sequencing of the corresponding clones have been used to guide peptide amino acid (aa) sequences. The observations are convincing if we consider the fact that peptides utilize well-known electrostatic, hydrogen bonding, hydrophobic, and van der Waals interactions to potentially recognize material surfaces possessing regularly distributed functional groups, as are commonly found in molecular-recognition mechanisms between biomolecules. Inorganic surfaces of metals, semiconductors, and metal oxides, and organic surfaces of carbon nanotubes, fullerenes, carbon nanohorns, and synthetic polymers are successful peptide targets. The resulting peptides have then been utilized as novel modifiers of nanomaterial surfaces and reduction catalysts for the production of inorganic nanoparticles. Polystyrene (PS), an all-purpose vinyl polymer frequently used for plastic plates in biological assays, attracts attention as a historical peptide target, even though PS has a simple chemical structure composed of an alkyl main chain and a lateral phenyl group joined to every other main-chain carbon. Kay et al. reported PS-specific 36and 22-mer sequence peptides isolated by the PD method, and suggested that Tyr and Trp enriched after biopanning might take part in p–p interactions with PS. However, their data showing whether the peptide absolutely recognized the positions either of the alkyl chains or of the phenyl group were unclear, because 1) some clones strongly bound to g-ray-irradiated PS, which would have surface carboxyl groups, 2) the strength of clone binding was dependent on blocking and washing with or without proteins (although these observations are certainly significant for practical applications of PS plates in biological assays), and 3) some clones similarly bound to polyvinyl chloride, which has a different chemical structure from PS. Accordingly, specific interactions between PS and peptides have remained a matter of debate. For successful isolation of polymer-recognizing peptides with high specificity and selectivity, we propose the importance of polymer stereoregularity, which can be an essential requirement for peptide targets. In fact, heptamer peptides that bind to film surfaces composed of isotactic (alpha carbon (Ca) atoms of adjacent monomer units have the same configuration) and syndiotactic (Ca atoms of adjacent monomer units have alternating configurations) poly(methyl methacrylate)s (PMMAs), but which do not bind to other reference stereoregular PMMAs, have already been isolated by the PD method. Therefore, an analysis of peptides that recognize stereoregular PS should provide an answer to our hypothesis. When stereoregular PS, with simpler structural components, is applied in the PD method, the potential of short peptides to recognize synthetic polymers should be clearer, so stereoregular PS should be the best peptide target. The work in this paper was aimed at isolating novel heptaACHTUNGTRENNUNGmer peptides that would show high specificity to syndiotactic PS (sPS) by the PD method (Figure 1). It is well known that sPS adopts TTGG helical conformations (T and G indicate trans and gauche conformations, respectively) when films are appropriately prepared with suitable solvents such as xylene and toluene. Evaporation of bulk solvents fabricates d form sPS films complexed with solvent molecules, while further evaporation of the complexed solvents fabricates empty d form (de) porous films. Here, the de form sPS was used as the peptide target. Since the regular assembly structure of target polymers offers the potential for augmentation of interactions with peptides, we expected that the peptide isolation should rationally be achievable. Atactic PS (aPS; a mixture of syndiotactic and isotactic sequences; Table 1), isotactic PS (iPS), and the d form

Biological selection of peptides for poly(l-lactide) substrates.

Short peptides that recognize the alpha form of poly( l-lactide) (PLLA) crystalline films were identified from a phage-displayed peptide library. An enzyme-linked immunosorbent assay (ELISA) revealed that the apparent binding constants of the phage clones for the alpha form of PLLA were greater than those of the unselected phage library. The specificity index for the alpha form of PLLA referred to a structurally similar atactic poly(methyl methacrylate) (at-PMMA), supporting the alpha form of PLLA specific binding of the selected phage. Amino acid residues with proton-donor lateral groups and hydrophobic alkyl groups were relatively enriched in a sequence of heptapeptides on the specific phage clones, thereby suggesting the presence of hydrogen bonding as well as hydrophobic interactions between the alpha form of PLLA and the peptides. Surface plasmon resonance (SPR) analysis revealed that the binding constant of the freed c22 heptapeptide (Gln-Leu-Met-His-Asp-Tyr-Arg) for the alpha form of PLLA was greater than those for reference at-PMMA, amorphous PLLA, and the beta form of PLLA. It was found that c22 peptide can recognize slight differences in PLLA polymorphs such as a crystalline state and an arrangement of PLLA functional groups.

Biological identification of peptides that specifically bind to poly(phenylene vinylene) surfaces: Recognition of the branched or linear structure of the conjugated polymer

Peptides that bind to poly(phenylene vinylene) (PPV) were identified by the phage display method. Aromatic amino acids were enriched in these peptide sequences, suggesting that a π-π interaction is the key interaction between the peptides and PPV. The surface plasmon resonance (SPR) experiments using chemically synthesized peptides demonstrated that the Hyp01 peptide, with the sequence His-Thr-Asp-Trp-Arg-Leu-Gly-Thr-Trp-His-His-Ser, showed an affinity constant (7.7 × 10(5) M(-1)) for the target, hyperbranched PPV (hypPPV) film. This value is 15-fold greater than its affinity for linear PPV (linPPV). In contrast, the peptide screened for linPPV (Lin01) showed the reverse specificity for linPPV. These results suggested that the Hyp01 and Lin01 peptides selectively recognized the linear or branched structure of PPVs. The Ala-scanning experiment, circular dichroism (CD) spectrometry, and molecular modeling of the Hyp01 peptide indicated that adequate location of two Trp residues by forming the polyproline type II (P(II)) helical conformation allowed the peptide to specifically interact with hypPPV.

Effect of Side-Chain Carbonyl Groups on the Interface of Vinyl Polymers with Water

The nature of the polymer-water interface in the poly(methyl 2-propenyl ether) (PMPE)-water model system is investigated by sum-frequency generation spectroscopy, which at the moment gives the best depth resolution among available techniques. PMPE, synthesized via living cationic polymerization, is structurally similar to poly(methyl methacrylate) (PMMA) except for lacking a carbonyl group. We here probe the polymer local conformation as well as the aggregation states of water at the interface. Comparing the results of our measurements to the PMMA-water system, the effect of a carbonyl group on the water structure at the interface is discussed. This knowledge should be crucial to the design and construction of highly functionalized polymer interfaces for bioapplications.

Effect of local chain dynamics on a bioinert interface.

Although many kinds of synthetic polymers have been investigated to construct blood-compatible materials, only a few have achieved success. To establish molecular designs for blood-compatible polymers, the chain structure and dynamics at the water interface must be understood using solid evidence as the first bench mark. Here we show that polymer dynamics at the water interface impacts on structure of the interfacial water, resulting in a change in protein adsorption and of platelet adhesion. As a particular material, a blend composed of poly(2-methoxyethyl acrylate) (PMEA) and poly(methyl methacrylate) was used. PMEA was segregated to the water interface. While the local conformation of PMEA at the water interface was insensitive to its molecular weight, the local dynamics became faster with decreasing molecular weight, resulting in a disturbance of the network structure of waters at the interface. This leads to the extreme suppression of protein adsorption and platelet adhesion.

Polymer-binding peptides for the noncovalent modification of polymer surfaces: Effects of peptide density on the subsequent immobilization of functional proteins

Peptides that specifically bind to polyetherimide (PEI) were selected, characterized, and used for the noncovalent modification of the PEI surface. The peptides were successfully identified from a phage-displayed peptide library. A chemically-synthesized peptide composed of the Thr-Gly-Ala-Asp-Leu-Asn-Thr sequence showed an extremely high binding constant for the PEI films (5.6 × 10(8) M(-1)), which was more than three orders of magnitude greater than that for the reference polystyrene films. The peptide was biotinylated and immobilized onto the PEI films to further immobilize streptavidin (SAv). The amount of SAv bound depended on the density of immobilized peptide. It gradually increased with an increasing density of immobilized peptide and achieved a maximum (2.1 pmol cm(-2)) at a peptide density of 19.8 pmol cm(-2). The ratio of peptide used for immobilizing SAv at the maximum value was only 11%, and was partially due to the low accessibility of SAv to the biotin moieties on the PEI films. Moreover, the amount of SAv bound gradually decreased at higher peptide densities, suggesting that the clustering of the peptides also inhibited the binding of SAv. Furthermore, peptides on the PEI films promoted the uniform immobilization of SAv with less structural denaturing. The immobilized SAv was able to further immobilize probe DNA to hybridize with its complementary DNA. These present results suggest that the density of immobilized peptide has a great impact on the surface modifications using polymer-binding peptides.

Binding Behavior of Lysine-Containing Helical Peptides to DNA Duplexes Immobilized on a 27 MHz Quartz-Crystal Microbalance

What role do electrostatic interactions play in the binding of a peptide to DNA? The binding behavior of eight lysine-containing peptides, which have differ in the number of lysine residues and their position in the peptide, has been investigated with the use of a quartz-crystal microbalance (see diagram).

Surface segregation of poly(2-methoxyethyl acrylate) in a mixture with poly(methyl methacrylate).

Poly(2-methoxyethyl acrylate) (PMEA) exhibits excellent blood compatibility. To understand why such a surface functionality exists, the surface of PMEA should be characterized in detail, structurally and dynamically, under not only ambient conditions, but also in water. However, a thin film of PMEA supported on a solid substrate can be easily broken, namely it is dewetted. Our strategy to overcome this difficulty is to mix PMEA with poly(methyl methacrylate) (PMMA). Differential scanning calorimetry and cloud point measurements revealed that the PMEA/PMMA blend has a phase diagram with a lower critical solution temperature. The blend surface was also characterized by X-ray photoelectron spectroscopy in conjunction with microscopic observations. Although PMEA is preferentially segregated over PMMA at the blend surface due to its lower surface free energy, the extent of segregation in the as-prepared films was not sufficient to cover the surface. Annealing the blend film at an appropriate temperature, higher than the glass transition temperature and lower than the phase-separation temperature of the blend, enabled us to prepare a stable and flat surface that was perfectly covered with PMEA.

Direct monitoring of allosteric recognition of type IIE restriction endonuclease EcoRII.

EcoRII is a homodimer with two domains consisting of a DNA-binding N terminus and a catalytic C terminus and recognizes two specific sequences on DNA. It shows a relatively complicated cleavage reaction in bulk solution. After binding to either recognition site, EcoRII cleaves the other recognition site of the same DNA (cis-binding) strand and/or the recognition site of the other DNA (trans-binding) strand. Although it is difficult to separate these two reactions in bulk solution, we could simply obtain the binding and cleavage kinetics of only the cis-binding by following the frequency (mass) changes of a DNA-immobilized quartz-crystal microbalance (QCM) responding to the addition of EcoRII in aqueous solution. We obtained the maximum binding amounts (Δmmax), the dissociation constants (Kd), the binding and dissociation rate constants (kon and koff), and the catalytic cleavage reaction rate constants (kcat) for wild-type EcoRII, the N-terminal-truncated form (EcoRII N-domain), and the mutant derivatives in its C-terminal domain (K263A and R330A). It was determined from the kinetic analyses that the N-domain, which covers the catalytic C-domain in the absence of DNA, preferentially binds to the one DNA recognition site while transforming EcoRII into an active form allosterically, and then the secondary C-domain binds to and cleaves the other recognition site of the DNA strand.

Effect of Mechanical Instability of Polymer Scaffolds on Cell Adhesion

The adhesion of fibroblast on polymer bilayers composed of a glassy polystyrene (PS) prepared on top of a rubbery polyisoprene (PI) was studied. Since the top PS layer is not build on a glassy, or firm, foundation, the system becomes mechanically unstable with decreasing thickness of the PS layer. When the PS film was thinner than 25 nm, the number of cells adhered to the surface decreased and the cells could not spread well. On a parallel experiment, the same cell adhesion behavior was observed on plasma-treated PS/PI bilayer films, where in this case, the surface was more hydrophilic than that of the intact films. In addition, the fluorescence microscopic observations revealed that the formation of F-actin filaments in fibroblasts attached to the thicker PS/PI bilayer films was greater than those using the thinner PS/PI bilayer films. On the other hand, the thickness dependence of the cell adhesion behavior was not observed for the PS monolayer films. Taking into account that the amount of adsorbed protein molecules evaluated by a quartz crystal microbalance method was independent of the PS layer thickness of the bilayer films, our results indicate that cells, unlike protein molecules, could sense a mechanical instability of the scaffold.