Substrate-Induced Conformation of an Artificial Receptor with Two

Nanoarchitectonics Research Center, National Institute of Advanced Industrial Science and Technology and CREST, JST (Japan Science and Technology), ...
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Langmuir 2002, 18, 813-818

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Substrate-Induced Conformation of an Artificial Receptor with Two Receptor Sites Masahiro Higuchi,*,† Tomoyuki Koga,† Kazuhiro Taguchi,† and Takatoshi Kinoshita‡ Nanoarchitectonics Research Center, National Institute of Advanced Industrial Science and Technology and CREST, JST (Japan Science and Technology), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan, and Department of Materials Science & Engineering, Nagoya Institute of Technology and CREST, JST (Japan Science and Technology), Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan Received August 16, 2001. In Final Form: November 5, 2001 Poly(γ-methyl L-glutamate)s with Asp, Tyr, Trp, and Ser residues at the amino terminal as the binding sites in the nicotinic acetylcoline receptor and γ-aminobutyric acid receptor, respectively, were prepared. The number average degree of polymerization of the polypeptides was 22. Dipalmitoylphosphatidylcholine monolayers containing the polypeptides were formed on buffered subphase solutions with and without substrates and were transferred onto gold-deposited glass plates. The polypeptides rearranged to form an assembly, owing to the interaction between the terminal amino acids and the substrates in the lipid monolayer on the buffer solution containing substrates. The polypeptide assembly vertically oriented to the monolayer surface. The binding ability of the substrates to the polypeptide assembly in the lipid monolayer was characterized by surface plasmon resonance measurements. The polypeptide assembly in the lipid monolayer conditioned on the acetylcholine aqueous solution exhibited a specific binding ability for the acetylcholine molecule. However, the polypeptide assembly lipid monolayer system prepared on the γ-aminobutyric acid aqueous solution showed a specific binding ability for the γ-aminobutyric acid. This behavior may have arisen from a substrates-induced rearrangement of the polypeptide assembly in the lipid monolayer, forming corresponding binding sites similar to that found in the nicotinic acetylcholine receptor and γ-aminobutyric acid receptor, respectively.

Introduction The cellular response to a particular extracellular signaling molecule depends on its binding to a specific receptor protein located on the surface of a target cell. The protein contains a unique distribution of functional groups, such as charged, hydrogen bonding, and hydrophobic amino acids, on its exterior surface. The specific location of these amino acids on the protein surface yields the specific binding site of the particular signaling molecule.1 In a nicotinic acetylcholine receptor, which is the longest known and best studied neuroreceptor, a specific arrangement of tyrosine and tryptophan residues of R-subunit, and tryptophan and asparatate residues of γ-subunit, form an acetylcholine binding site.2-5 However, antibodies can recognize a great number of molecules owing to highly variable amino acid sequences in the hypervariable region in those binding sites.6,7 Studies on synthetic molecular recognition systems having an ordered assembly of functional groups in the binding site may be important not only to contribute to the understanding of * To whom correspondence should be addressed. † National Institute of Advanced Industrial Science and Technology and CREST, JST (Japan Science and Technology). ‡ Nagoya Institute of Technology and CREST, JST (Japan Science and Technology). (1) Lodish, H.; Baltimore, D.; Berk, A.; Zipursky, S. J.; Matsudaira, P.; Darnell, J. Molecular Cell Biology; Scientific American Books: New York, 1995. (2) Unwin, N. Nature 1995, 373, 37. (3) Galzi, J.-L.; Changeus, J.-P. Neuropharmacology 1995, 34, 536. (4) Karlin, A.; Akabas, M. H. Neuron 1995, 15, 1231. (5) Zhong, W.; Gallivan, J. P.; Zhang, Y.; Li, L.; Lester, H. A.; Dougherty, D. A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 12088. (6) Edmundson, A. B.; Ely, K. R.; Abola, E. E.; Schiffer, M.; Panagiotpoulos, N. Biochemistry 1975, 14, 3953. (7) Schultz, P. G. Science 1988, 240, 426.

a simple and/or essential mechanism for signal reception and transduction through biological interfaces but also to provide the basis of a molecular device capable of receiving and transferring information. It has been reported that artificial receptors composed of the ordered assembly of functional groups exhibit a specific binding ability of a particular substrate. For example, Stewart et al.8 reported on the receptor consisting of four parallel amphiphilic helices, whose free amino terminals have Ser, His, Asp, and Glu as a model of chymotrypsin. The receptor has an affinity for chymotrypsin ester substrates similar to that of chymotrypsin and hydrolyzes them in a manner similar to chymotrypsin. Hamilton et al.9 reported on a family of synthetic receptors composed of four peptide loops arrayed around a central calixarene core. They suggested that a range of differently functionalized receptors can be prepared by varying the sequence of the loop regions. In this paper, we propose a novel approach to preparing artificial receptors consisting of a polypeptide assembly in a lipid monolayer. In our system, the polypeptides can rearrange in the lipid monolayer owing to the interaction between the polypeptides and the substrate, because the polypeptides are not fixed by covalent bonds in the assembly. Our concept of the formation of a substrate-induced recognition structure in a polypeptide assembly is outlined in Figure 1. The polypeptides used for the nicotinic acetylcholine receptor model are poly(γ-methyl L-glutamate)s having Tyr, Trp, and Asp at the amino terminal, respectively. For the γ-aminobutyric acid receptor, Galves et al.10 reported that the Ser and Tyr residues were found to be critical for γ-aminobutyric acid binding (8) Hahn, K. W.; Klis, W. A.; Stewart, J. M. Science 1990, 248, 1544. (9) Park, H. S.; Lin, Q.; Hamilton, A. D. J. Am. Chem. Soc. 1999, 121, 8.

10.1021/la011305+ CCC: $22.00 © 2002 American Chemical Society Published on Web 01/10/2002

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Figure 1. Preparation of a recognition structure composed of a polypeptide assembly induced by interaction with a substrate in a lipid monolayer.

by the point mutation technique. We used the poly(γmethyl L-glutamate)s having Ser and Tyr at the amino terminal, respectively, for the model polypeptides for the γ-aminobutyric acid receptor. These polypeptides form a corresponding assembly to the added substrate, acetylcholine or γ-aminobutyric acid, owing to an interaction between the substrate and the amino terminal functional groups of the polypeptides in a lipid monolayer at the air-water interface. That is to say, acetylcholine induces the formation of a polypeptide assembly containing the polypeptides having Tyr, Trp, and Asp at the amino terminals, whereas the assembly containing the polypeptides having Ser and Tyr at the amino terminals is induced by γ-aminobutyric acid. The substrate-induced polypeptide assembly immobilized on the gold-deposited glass plate acted as a specific receptor of the corresponding substrate. Preliminary results of the substrate-induced formation of a recognition structure in a polypeptide assembly at an air-water interface have been reported previously.11 Experimental Section Materials. Poly(γ-methyl L-glutamate) (s(Tri)-PMG)) was obtained by the polymerization 12 of the N-carboxyanhydride of the L-glutamic acid γ-methyl ester13 in dimethylformamide (DMF) solution with 2-aminoethane-S-tritylthiol as the initiator. The molar ratio of anhydride to the initiator was 30, and the polymerization occurred at room temperature over 24 h. The (10) Galvez, T.; Parmentier, M.-L.; Joly, C.; Malitschek, B.; Kaupmann, K.; Kuhn, R.; Bittiger, H.; Froestl, W.; Bettler, B.; Pin, J.-P. J. Biol. Chem. 1999, 274, 13362. (11) Higuchi, M.; Taguchi, K.; Wright, J. P.; Kinoshita, T. J. Polym. Sci., Part A: Polym. Chem. 2000, 38, 2186. (12) Bamford, C. H.; Elliot, A.; Hanby, W. E. Synthetic Polypeptides; Academic Press: New York, 1956. (13) Daly, W. H.; Poche, D. Tetrahedron Lett. 1988, 29, 5859.

DMF solution was poured into ethanol, and the precipitated s(Tri)-PMG was purified by reprecipitation from DMF into ethanol until no unreacted 2-aminoethane-S-tritylthiol was detected spectroscopically. A number average molecular weight of 3400 was estimated from the molar ratio of the trityl moiety to the γ-methyl L-glutamate residues of s(Tri)-PMG. The ratio was determined from the absorbance at 225 nm of the DMF solution of the s(Tri)-PMG on the basis of the molar extinction coefficient of 2-aminoethane-S-tritylthiol. s(Tri)-PMG that had Fmoc-L-Asp(OBut) at the amino terminal was obtained from the coupling reaction between s(Tri)-PMG and Fmoc-L-Asp(OBut) in the DMF solution for 24 h with 1-ethyl3(3-dimethylaminopropyl)carbodiimide hydrochloride and Nhydroxybenzotriazole.14 The amino terminal Fmoc protecting group of the polypeptide was removed in the DMF solution containing 20 vol % piperidine for 30 min. We obtained the polypeptide that had the Asp residue at the amino terminal, and the thiol group at the carboxyl terminal (s-PMG(Asp)), by removing the tert-butyl protecting group of the amino terminal Asp residue, and the trityl protecting group of the carboxyl terminal thiol moiety in a trifluoroacetic acid solution containing 2.5 vol % water and 2.5 vol % 1,2-ethanedithiol, at room temperature for 1.5 h. Polypeptides that had Tyr, Trp, and Ser residues at the amino terminal (s-PMG(Tyr), s-PMG(Trp), and s-PMG(Ser)) were obtained in a similar manner as above, using Fmoc-L-Tyr(But), Fmoc-L-Trp, and Fmoc-L-Ser(But), respectively (Chart 1). The degrees of introduction of Asp, Tyr, Trp, and Ser residues to the amino terminal of the polypeptide were determined to be 75, 71, 70, and 76%, respectively, from the fluorescence intensity of the Fmoc protecting group at 310 nm in the DMF solution. Furthermore, the poly(γ-methyl Lglutamate) that had its thiol group at the carboxyl terminal (sPMG) was obtained by removing the trityl protecting group of the carboxyl terminal thiol moiety of s(Tri)-PMG in a similar manner. (14) Houben, J. F.; Fissi, A.; Bacciola, D.; Rosato, N.; Pieroni, O.; Ciardelli, F. J. Biol. Macromol. 1983, 5, 94.

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Chart 1. Chemical Structure of s-PMG(Asp), s-PMG(Tyr), s-PMG(Trp), and s-PMG

Surface Pressure-Area Measurements. Surface pressure-area (π-A) isotherms of a dipalmitoylphosphatidylcholine (DPPC) monolayer containing the polypeptides were measured with a multitrough type Langmuir film balance (Nippon Laser & Electronics Lab., NL-LB-200-MTC). The subphase water used was Milli-Q treated and doubly distilled water. DPPC and the polypeptides were each dissolved in DMF. These solutions were mixed and poured into a glass flask, forming a thin film on the inner surface of the flask after solvent evaporation. Spectroscopic grade chloroform was added to the flask to dissolve the thin film. The concentration of DPPC was 0.2 mg/mL. The molar ratio of the polypeptide to DPPC was 0.01. A measured small amount of the solution was delivered to the water surface from a Termo microsyringe. π-A isotherms of the monolayer on a buffer solution (50 mM HEPES-NaOH, pH 7.2) and on a buffer solution containing 0.1 mM substrate were taken at a compression rate of 5 mm/min and a subphase temperature of 25 °C, respectively. The substrate-induced surface area changes of the DPPC monolayer containing the polypeptides at a constant surface pressure were measured as follows. The monolayer on the buffer solution was compressed to the desired surface pressure, and then the surface area of the monolayer was kept constant while the monolayer was transferred from the buffer surface to the substrate solution surface at 50 mm/min. The changes of the surface area on the substrate solution were recorded continuously at the desired surface pressure. Spectroscopic Measurement. The circular dichroism (CD) spectra of the polypeptides in the trifluoroethanol and DPPC monolayer were measured with a JASCO J-720WI CD spectrometer. Four layers of the DPPC monolayers containing the polypeptides were transferred onto a quartz plate by a vertical dipping method at a surface pressure of 40 mN/m. The Fourier transform infrared reflection-absorption spectra (FTIR-RAS) of the DPPC monolayers containing the polypeptides were measured with a Perkin-Elmer Spectrum 2000 equipped with a PIKE, 80Spec reflectance accessory. The monolayer transfer onto a gold-deposited glass plate was carried out using the vertical dipping method, with the monolayers attaching to the plate on the upstroke, at surface pressures of 15 and 40 mN/m, respectively. The 1700-1500 cm-1 regions of the spectra were analyzed as a sum of the Gaussian/Lorentzian composition of individual bands. When the ratio of the Gaussian/ Lorentzian was 9/1, the sum of the calculated individual bands was best fit to the experimental spectra. The molecular orientation of the polypeptides in the DPPC monolayer was assessed according to the ratio method using FTIR-RAS. By comparison of the theoretical values of the ratio of the amide I and II absorbencies with the experimental values of FTIR-RAS, the tilt angle of the helix axis from the surface normal was determined.15-17 Surface Plasmon Resonance Measurements. The binding behavior of the substrates, acetylcholine (ACh), and γ-aminobutyric acid (GABA) toward the polypeptide assembly in the DPPC monolayer on the gold-deposited glass plate was measured using surface plasmon resonance (SPR) spectroscopy (Nippon Laser & Electronics Lab., SPR670B). The DPPC monolayers containing the polypeptides were transferred onto the gold-deposited glass plates by a horizontal lifting method from the buffer solution (15) Fringeli, U. P.; Gunthard, H. H. Membrane Spectroscopy; Springer-Verlag: Belin and Heidelberg, Germany, 1981. (16) Gremlich, H. U.; Fringeli, U. P.; Schwyzer, R. Biochemistry 1983, 22, 4257. (17) Boncheva, M.; Vogel, H. Biophys. J. 1997, 73, 1956.

Figure 2. Circular dichroism spectra of s-PMG(Asp), s-PMG(Tyr), s-PMG(Trp), and s-PMG in (a) trifluoroethanol and in (b) a DPPC monolayer. The molar ratio of the polypeptides to the DPPC was 0.01. The molar ratio of s-PMG(Asp), s-PMG(Tyr), s-PMG(Trp), and s-PMG was 1:1:1:3. The four monolayers were transferred onto the quartz plate at 40 mN/m by the vertical dipping method. and from the buffer solution containing the 0.1 mM substrate, respectively. The surface pressure of the monolayers was maintained at 40 mN/m during the transfer. The LangmuirBlodgett (LB) films prepared on the buffer solution containing 0.1 mM substrate were immersed in pure buffer solution overnight to remove the absorbed substrate on the surface. Changes in the refractive index of the medium near the gold surface were measured using SPR.18 A plot of reflected intensity versus the angle of incidence shows a minimum, θm, corresponding to the excitation of the surface plasmons at the gold-solution interface. The value θm shifts with changes in the refractive index of the interfacial region near the gold surface.19-21 These changes in shift, ∆θm, are proportional to the amount of binding substrate at the surface. During the measurement, 50 mM HEPES-NaOH buffer, pH 7.2, was passed over the sample surface at 10 µL/min. This flow was periodically replaced with the buffer solution containing the substrate at increasing concentration, while the temperature was kept constant at 28 °C.

Results and Discussion Structure of Polypeptides in the DPPC Monolayer. The secondary structure of the polypeptides was estimated from the CD spectra. In the trifluoroethanol solution, the CD spectrum of s-PMG(Asp), s-PMG(Tyr), s-PMG(Trp) as the nAChR model, and s-PMG showed a double minimum profile at 208 and 222 nm, typical of a stable, right-handed R-helix 22 (Figure 2a). The molar ratio of s-PMG(Asp), s-PMG(Tyr), s-PMG(Trp), and s-PMG was fixed to 1:1:1:3. However, the CD spectrum of the polypeptides in the DPPC monolayer, which was transferred onto a quartz plate from the buffer solution, showed a decrease in the molar ellipticity at 208 nm (Figure 2b). This distortion of the spectrum implies that the R-helical (18) Otto, A. Z. Phys. 1968, 216, 398. (19) Eagen, C. F.; Weber, W. H. Phys. Rev. B 1979, 19, 5068. (20) Nylander, C.; Liedberg, B.; Lind, T. Sens. Actuators 1982/83, 3, 79. (21) Knoll, W. Annu. Rev. Phys. Chem. 1998, 49, 569. (22) Greenfield, N.; Fasman, G. Biochemistry 1969, 8, 4018.

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Figure 3. Surface pressure-area isotherms of DPPC monolayers containing s-PMG(Asp), s-PMG(Tyr), s-PMG(Trp), and s-PMG on buffered subphase solutions (a) with and (b) without 0.1 mM at 25 °C. The molar ratio of the polypeptides to DPPC was 0.01. The molar ratio of s-PMG(Asp), s-PMG(Tyr), s-PMG(Trp), and s-PMG was 1:1:1:3.

rods form an assembly in the membrane.23-25 Furthermore, we reported in the previous paper that the poly(γ-methyl L-glutamate)s formed a self-assembled bundle in a solid-state lipid monolayer, as determined by atomic force microscopic observations.11,26 We investigated the ACh-induced structural changes of the DPPC monolayer containing s-PMG(Asp), s-PMG(Tyr), s-PMG(Trp), and s-PMG by the π-A isotherm and FTIR-RAS measurements. Figure 3 shows the π-A isotherms of the DPPC monolayer containing these polypeptides on the buffered subphase solutions with and without 0.1 mM ACh, conducted at a constant temperature of 25 °C. The abscissa indicates the area per DPPC molecule. The π-A isotherm of the monolayer on the buffer solution containing 0.1 mM ACh displayed a significant condensation at the phase transition region around 15 mN/m. To elucidate the effect of ACh on the structural changes of the polypeptides in the monolayer, the AChinduced surface area changes of the monolayer were investigated under a constant surface pressure of 15 ( 0.1 mN/m. Figure 4 shows the surface area changes of the monolayer after the monolayer was transferred from the buffer surface to the ACh solution surface and from the ACh solution surface to the buffer surface, respectively. At the air-ACh solution interface, the monolayer immediately shrank to the equilibrium surface area within ca. 10 min. However, after the monolayer transferred from the ACh solution surface to the buffer surface, it gradually expanded. The ACh-induced surface area changes were not observed for the pure DPPC monolayer. This result suggests that the polypeptides rearrange to form a compact, high-order structure owing to the interaction between the ACh and terminal amino acids of the polypeptides. The DPPC monolayer containing s-PMG(Asp), s-PMG(Tyr), s-PMG(Trp), and s-PMG was transferred vertically onto a gold-deposited glass plate from the subphase to the (23) Maeda, H.; Hato, H.; Ikeda, S. Biopolymers 1984, 23, 1333. (24) Rosenheck, K.; Schneider, A. S. Proc. Natl. Acad. Sci. U.S.A., 1973, 70, 3458. (25) Pieroni, O,; Fissi, A.; Houben, J. L.; Ciardelli, F. J. Am. Chem. Soc. 1985, 107, 2990. (26) Higuchi, M.; Minoura, N.; Kinoshita, T. Langmuir 1997, 13, 1616.

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Figure 4. Changes in the monolayer area of the DPPC monolayer containing s-PMG(Asp), s-PMG(Tyr), s-PMG(Trp), and s-PMG after the monolayer was transferred from the buffer surface to the ACh solution surface and from the ACh solution surface to the buffer surface, respectively. The transfer of the monolayer was carried out at 50 mm/min. The molar ratio of the polypeptides to DPPC was 0.01. The molar ratio of s-PMG(Asp), s-PMG(Tyr), s-PMG(Trp), and s-PMG was 1:1:1:3.

Figure 5. FTIR-RAS of DPPC monolayers containing s-PMG(Asp), s-PMG(Tyr), s-PMG(Trp), and s-PMG transferred onto a gold-deposited glass plate from the buffer solution (a) at 15 mN/m, and (b) at 40 mN/m, and from the buffer solution containing 0.1 mM ACh (c) at 15 mN/m and (d) 40 mN/m. The molar ratio of the polypeptides to DPPC was 0.01. The molar ratio of s-PMG(Asp), s-PMG(Tyr), s-PMG(Trp), and s-PMG was 1:1:1:3.

air phase both on the buffer solution and the buffer solution containing 0.1 mM ACh at 15 mN/m and 40 mN/m, respectively. The orientation of the polypeptides in the monolayer was analyzed by FTIR-RAS (Figure 5). The spectra of the monolayers showed typical amide I bands at 1658 cm-1 (R-helix) and 1630 cm-1 (β-sheet), and amide II bands at 1550 cm-1 (R-helix) and 1530 cm-1 (β-sheet).27 It is indicated from the peak deconvolution that the polypeptides in the monolayer were mainly in the R-helix (27) Miyazawa, T.; Blout, E. R. J. Am. Chem. Soc. 1961, 83, 712.

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Table 1. Tilt Angle of the Polypeptide in the DPPC Monolayer Prepared on Various Subphase Solutions subphase buffer solution buffer solution with 0.1 mM ACh

surface pressure/mN m-1

tilt angle/deg

15 40 15 40

58 38 39 37

conformation containing the β-sheet structure. The helical content was estimated to be 69% from the molar ellipticity (Figure 2b). The tilt angle of the R-helix axis from the surface normal was estimated from the ratio of the individual intensities of the amide I to amide II band of the R-helix. The monolayer transferred from the buffer solution at 15 mN/m, and the intensity of amide I band was smaller than that of the amide II band (Figure 5a). However, in the monolayer transferred at 40 mN/m, the amide I band was larger than the amide II band (Figure 5b). This implies that the polypeptides aligned nearly parallel to the monolayer surface at 15 mN/m and that the polypeptides vertically oriented in the monolayer at 40 mN/m. In contrast, both monolayers which transferred from the buffered subphase with ACh at 15 mN/m (Figure 5c) and at 40 mN/m (Figure 5d) showed a larger intensity of the amide I band than that of the amide II band. The tilt angles of the polypeptides in the DPPC monolayer are summarized in Table 1. These results suggest that the polypeptides change orientation from the parallel to the perpendicular to the membrane surface to form the assembly as a result of the interaction between the aqueous ACh and the amino terminal groups of the polypeptides in the air-water interface. Substrate Recognition Properties of the DPPC Monolayers Containing Polypeptides. We investigated the binding ability of the ACh molecules to the DPPC monolayers containing s-PMG(Asp), s-PMG(Tyr), and s-PMG(Trp) as the model polypeptides of nAChR and s-PMG by SPR measurements. The monolayers containing the polypeptides were transferred onto the gold-deposited glass plates by the horizontal lifting method from the buffer solution and from the buffer solution containing the 0.1 mM ACh, respectively. The structure of the polypeptide assembly in the monolayer was immobilized on the gold-deposited glass plate by conjugation of the carboxyl terminal thiol of the polypeptides to the gold surface. The polypeptide assembly obtained on the gold surface was stable, and the buffer flow did not cause it to be removed from the monolayer. Figure 6 shows the kinetics for the binding of ACh to the monolayer which was transferred from the buffer solution with and without 0.1 mM ACh, respectively, when the flow passed over the monolayer was replaced with the buffer solution containing 2.19 µM ACh. The introduction of ACh into the monolayer prepared on the buffer induced relatively small shifts in the resonance angle of the surface plasmon, ∆θm. However, the introduction of ACh into the monolayer prepared on the buffer containing 0.1 mM ACh led to a large shift in ∆θm. Furthermore, the value of ∆θm did not decrease after the flow which passed over the monolayer was again replaced with the buffer solution within the measurement period. Figure 7 shows the isotherms of ACh binding to the monolayers prepared on the buffer solution with and without 0.1 mM ACh, respectively. The isotherm for Ach, which binds with the monolayer prepared on the ACh solution, exhibits a typical, Langmuir-type saturation behavior. This indicates that the monolayer provides a specific binding site for the ACh molecules.

Figure 6. Binding kinetics of the ACh molecule to the DPPC monolayer containing s-PMG(Asp), s-PMG(Tyr), s-PMG(Trp), and s-PMG prepared on the buffer solution with and without 0.1 ACh. The concentration of ACh molecules introduced to the monolayer surface was 2.16 µM. The molar ratio of the polypeptides to DPPC was 0.01. The molar ratio of s-PMG(Asp), s-PMG(Tyr), s-PMG(Trp), and s-PMG was 1:1:1:3.

Figure 7. Binding isotherms of the ACh molecule to the DPPC monolayer containing s-PMG(Asp), s-PMG(Tyr), s-PMG(Trp), and s-PMG prepared on the buffer solution with and without 0.1 ACh.

The equilibrium between the binding and the dissociation of the ACh molecules to the polypeptide assembly in the monolayer can be represented by28 kbind

[polypeptide assembly] + [ACh] {\ } k diss

[polypeptide assembly-ACh] (1) where kbind and kdiss represent the rate of binding and the dissociation of the ACh molecules to the polypeptide assembly in the monolayer, respectively. The binding constant, K, is denoted by

K ) kbind/kdiss

(2)

Shift changes in the surface plasmon resonance angle, ∆θm, for the monolayer, based on the introduction of the ACh molecules to the monolayer surface, are represented by (28) Higuchi, M.; Wright, J. P.; Taguchi, K.; Kinoshita, T. Langmuir 2000, 16, 7061.

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Figure 8. Binding isotherms of the ACh and GABA molecule to the DPPC monolayer containing s-PMG(Asp), s-PMG(Tyr), s-PMG(Trp), s-PMG(Ser), and s-PMG prepared on the buffer solution (a) with 0.1 ACh and (b) with 0.1 mM GABA. The molar ratio of the polypeptides to DPPC was 0.01. The molar ratio of s-PMG(Asp), s-PMG(Tyr), s-PMG(Trp), and s-PMG was 1:1:1:4.

d(∆θm)/dt ) kbind[ACh]∆θm max - (kbind[ACh] + kdiss)∆θm (3) where ∆θm max represents the saturated value of ∆θm, obtained from Figure 7, when the binding site on the monolayer is completely occupied by the ACh molecules. The binding constants, K (calculated using eqs 2 and 3), for the binding of ACh to the monolayer which was prepared on the buffer solution with 0.1 mM ACh was 2.66 × 105 M. This value was 13 times larger than that of the monolayer prepared on the buffer solution without ACh (1.96 × 104 M). This result suggests that the polypeptide assembly, which was formed by the interaction between ACh and the amino terminal amino acids (Asp, Tyr, and Trp) of the polypeptides in the DPPC monolayer, act as a specific binding site similar to that found in the nicotinic acetylcholine. Diverse Recognition Ability of the DPPC Monolayers Containing Polypeptides. We demonstrated a diverse recognition ability of the DPPC monolayer containing polypeptides. The DPPC monolayers containing s-PMG(Asp), s-PMG(Tyr), and s-PMG(Trp) as the model polypeptides of nAChR, and s-PMG(Tyr) and s-PMG(ser) as the model polypeptides of GAGAR and s-PMG, were transferred onto gold-deposited glass plates by the horizontal lifting method and immobilized on the surface by the buffer solution containing 0.1 mM ACh and 0.1 mM GABA, respectively. The molar ratio of s-PMG(Asp), s-PMG(Tyr), s-PMG(Trp), s-PMG(Ser), and s-PMG was fixed at 1:1:1:1:4. Figure 8 shows the binding isotherms of ACh and GABA to the monolayers prepared on the buffer solution (a) with 0.1 mM ACh and (b) with 0.1 mM GABA, respectively. The membrane prepared on the ACh solution showed specific binding ability to the ACh compared with

Table 2. Binding Constant of Analytes to the Polypeptide Assembly in DPPC Monolayer Prepared on Various Subphase Solutions subphase buffer solution with 0.1 mM ACh buffer solution with 0.1 mM GABA

analyte

binding constant/M-1

ACh GABA ACh GABA

1.54 × 105 3.62 × 104 4.06 × 104 7.58 × 104

the GABA. However, the membrane prepared on the GABA solution exhibited specific binding ability to the GABA. In Table 2, the binding constants of ACh and GABA to the monolayer prepared on the buffer solution with 0.1 mM ACh and 0.1 mM GABA are summarized. This result suggests that the polypeptides in the DPPC monolayer rearrange to form the binding site corresponding to the added substrate in the subphase. That is to say, the monolayer composed of the same components (s-PMG(Asp), s-PMG(Tyr), s-PMG(Trp), and s-PMG(Ser)) could form a receptor with diversed recognition ability owing to the rearrangement of the polypeptide induced by added substrate in the subphase. In conclusion, the polypeptides having various amino acids at the amino terminal formed a corresponding assembly to the added aqueous substrate in the DPPC monolayer owing to the interaction between the substrate and amino terminal functional groups of the polypeptides. The substrate-induced polypeptide assembly acted as the specific receptor of the corresponding substrate. This system is an example of a mimetic system of the antibodylike diversity of recognition. LA011305+