Highly Specific Affinities of Short Peptides against Synthetic Polymers

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Highly Specific Affinities of Short Peptides against Synthetic Polymers Takeshi Serizawa,*,†,‡ Toshiki Sawada,†,§ and Hisao Matsuno| Research Center for AdVanced Science and Technology (RCAST), The UniVersity of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan, Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan, DiVision of Materials Science and Engineering, Shibaura Institute of Technology (SIT), 3-7-5 Toyosu, Koto-ku, Tokyo 135-8548, Japan, and Komaba Open Laboratory (KOL), The UniVersity of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan ReceiVed June 21, 2007. In Final Form: August 16, 2007 We investigated polymer-binding 7-mer peptides that recognize differences in the polymer stereoregularity of all-purpose poly(methyl methacrylate)s (PMMAs) with simple chemical structures. Quantitative surface plasmon resonance measurements detected association/dissociation processes of the peptides against PMMA film surfaces, followed by an estimation of kinetic parameters such as association/dissociation rate constants and affinity constants. Greater association and smaller dissociation constants of the peptides were observed against a target isotactic PMMA than the structurally similar reference syndiotactic PMMA, followed by greater affinity constants against the target. A c02 peptide composed of the Glu-Leu-Trp-Arg-Pro-Thr-Arg sequence showed the greatest affinity constant (2.8 × 105 M-1) for the target, which was 41-fold greater than that for the reference, thus demonstrating extremely high peptide specificities. The substitution of each amino acid of the c02 peptide to Ala (Ala scanning) clearly revealed the essential amino acids for the affinity constants; the essential order was Pro5 . Thr6 > Arg7 > Glu1 > Arg4. In fact, the shorter 4-mer peptide composed of the C-terminal Arg-Pro-Thr-Arg sequence of the c02 peptide still demonstrated strong target specificity, although the N-terminal 4-mer peptide Glu-Leu-Trp-Arg completely lost its specificity. The possible conformations modeled with Molecular Mechanics supported the significance of the ArgPro-Thr-Arg sequence. The thermodynamic parameters of the c02 peptide suggested an induced fit mechanism for the specific affinity. The present affinity analyses of polymer-recognizing peptides revealed significant and general information that was essential for potential applications in peptidyl nanomaterials.

Introduction Over the past decade, peptide motifs that specifically bind to artificial materials have been revealed by selection from genetically engineered cell-surface display (CSD) and phage display (PD) peptide libraries with high diversities.1 Inorganic surfaces of metals,2 semiconductors,3 and metal oxides4 as well as organic molecules such as carbon nanotubes,5 carbon nanohorns,6 fullerenes,7 and synthetic polymers8 have been used as peptide targets. The resulting peptides have the potential as novel peptidyl nanomaterials such as catalysts for the preparation of inorganic nanoparticles,9 adsorbents for patterning,2c surface †

RCAST, The University of Tokyo. PRESTO, JST. § Division of Materials Science and Engineering, SIT. | KOL, The University of Tokyo. ‡

(1) (a) Sarikaya, M.; Tamerler, C.; Jen, A. K.-Y.; Schulten, K.; Baneyx, F. Nat. Mater. 2003, 2, 577. (b) Sarikaya, M.; Tamerler, C.; Schwartz, D. T.; Baneyx, F. Annu. ReV. Mater. Res. 2004, 34, 373. (2) (a) Brown, S. Nat. Biotechnol. 1997, 15, 269. (b) Brown, S.; Sarikaya, M.; Johnson, E. J. Mol. Biol. 2000, 299, 725. (c) Naik, R. R.; Stinger, S. J.; Agarwal, G.; Jones, S. E.; Stone, M. O. Nat. Mater. 2002, 1, 169. (3) (a) Whaley, S. R.; English, D. S.; Hu, E. L.; Barbara, P. F.; Belcher, A. M. Nature 2000, 405, 665. (b) Lee, S.-W.; Mao, C.; Flynn, C. E.; Belcher, A. M. Science 2002, 296, 892. (4) (a) Brown, S. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 8651. (b) Barbas, C. F., III; Rosenblum, J. S.; Lerner, R. A. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 6385. (c) Sano, K.-I.; Shiba, K. J. Am. Chem. Soc. 2003, 125, 14234. (d) Hayashi, T.; Sano, K.-I.; Shiba, K.; Kumashiro, Y.; Iwahori, K.; Yamashita, I.; Hara, M. Nano Lett. 2006, 6, 515. (5) (a) Wang, S.; Humphreys, E. S.; Chung, S.-Y.; Delduco, D. F.; Lustig, S. R.; Wang, H.; Parker, K. N.; Rizzo, N. W.; Subramoney, S.; Chiang, Y.-M.; Jagota, A. Nat. Mater. 2003, 2, 196. (b) Li, X.; Chen, W.; Zhan, Q.; Dai, L.; Sowards, L.; Pender, M.; Naik, R. R. J. Phys. Chem. B 2006, 110, 12621. (6) Kase, D.; Kulp, J. L., III; Yudasaka, M.; Evans, J. S.; Iijima, S.; Shiba, K. Langmuir 2004, 20, 8939. (7) Morita, Y.; Ohsugi, T.; Iwasa, Y.; Tamiya, E. J. Mol. Catal. B: Enzym. 2004, 28, 185.

modifiers,8c,9a,10 and modifiers of phages3b/proteins11 used for assembly. To further produce sophisticated functionalities for these peptides, it is necessary to quantitatively understand their strengths, specificities, and selectivities and the association/ dissociation rates of peptides directed against target substrates. To demonstrate the peptide affinities against target materials after the peptide selection process, enzyme-linked immunosorbent assays (ELISAs) using cell and phage clones displaying the corresponding peptides are commonly used. Although ELISA can estimate the relative amounts of clones bound to material surfaces, it is impossible to directly quantify the peptide affinities because (i) clones with high molecular weights (for example, the molecular weight of a M13 bacteriophage is approximately 16 300 00012) might adsorb onto material surfaces nonspecifically, (ii) plural peptide copies displayed on cells and phages might strengthen their affinities for targets through multiple interactions, and (iii) the displayed peptides that are covalently bound to the (8) (a) Adey, N. B.; Mataragnon, A. H.; Rider, J. E.; Carter, J. M.; Kay, B. K. Gene 1995, 156, 27. (b) Berglund, J.; Lindbladh, C.; Nicholls, I. A.; Mosbach, K. Anal. Commun. 1998, 35, 3. (c) Sanghvi, A. B.; Miller, K. P.-H.; Belcher, A. M.; Schmidt, C. E. Nat. Mater. 2005, 4, 496. (d) Serizawa, T.; Sawada, T.; Matsuno, H.; Matsubara, T.; Sato, T. J. Am. Chem. Soc. 2005, 127, 13780. (e) Serizawa, T.; Sawada, T.; Kitayama, T. Angew. Chem., Int. Ed. 2007, 46, 723. (f) Serizawa, T.; Techawanitchai, P.; Matsuno, H. ChemBioChem 2007, 8, 989. (9) (a) Slocik, J. M.; Stone, M. O.; Naik, R. R. Small 2005, 1, 1048. (b) Sano, K.-I.; Sasaki, H.; Shiba, K. Langmuir 2005, 21, 3090. (c) Ahmad, G.; Dickerson, M. B.; Church, B. C.; Cai, Y.; Jones, S. E.; Naik, R. R.; King, J. S.; Summers, C. J.; Kro¨ger, N.; Sandhage, K. H. AdV. Mater. 2006, 18, 1759. (d) Slocik, J. M.; Naik, R. R. AdV. Mater. 2006, 18, 1988. (10) (a) Pender, M. J.; Sowards, L. A.; Hartgerink, J. D.; Stone, M. O.; Naik, R. R. Nano Lett. 2006, 6, 40. (b) Sano, K.-I.; Sasaki, H.; Shiba, K. J. Am. Chem. Soc. 2006, 128, 1717. (c) Matsui, T.; Matsukawa, N.; Iwahori, K.; Sano, K.-I.; Shiba, K.; Yamashita, I. Langmuir 2007, 23, 1615. (11) Sano, K.-I.; Ajima, K.; Iwahori, K.; Yudasaka, M.; Iijima, S.; Yamashita, I.; Shiba, K. Small 2005, 1, 826. (12) Barbas, C. F., III; Burton, D. R.; Scott, J. K.; Silverman, G. J. Phage Display: A Laboratory Manual; Cold Spring Harbor Press: New York, 2001.

10.1021/la701822n CCC: $37.00 © 2007 American Chemical Society Published on Web 10/02/2007

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surface proteins of cells and phages might have a stable conformation suitable for target affinities through intramolecular interactions. Quartz crystal microbalance (QCM) measurements using whole phage clones, which determine the mass and/or elasticity of phages bound onto QCM electrodes, is a good potential method;4c,13 unfortunately, it is difficult to quantitatively interpret the data. Accordingly, it is necessary to directly analyze the affinities of peptides free from cell and phage bodies. Recently, interaction force analyses using scanning probe microscopy have been applied to isolated peptides,4d,5b,8c which were conjugated onto a cantilever tip, to directly verify the strength and specificity of this interaction. In fact, greater forces of isolated 12-mer peptides have been detected against target chlorine-doped polypyrrole,8c titanium oxide,4d and carbon nanotube5b surfaces. QCM measurements have also revealed the adsorption isotherms of the peptides and the affinity kinetics of proteins displaying multiple peptides against titanium oxide surfaces.11 More recently, the binding affinities of 42-mer tandem peptides with three repeating units against gold surfaces were kinetically analyzed by surface plasmon resonance (SPR) and QCM measurements, and these measurements suggested the possibility that both analytical methods detected different binding processes.14 These observations clearly suggest that peptides isolated by the CSD and PD methods undoubtedly show target specificities. However, detailed information on their affinity constants and kinetics, especially of extremely short peptides composed of less than 10 amino acids (aa) against target surfaces as well as structurally similar references, is still limited. Shorter material-recognizing peptides have the advantage of further chemical modification of these peptides. We are interested in short peptides that recognize structural differences in synthetic polymer film surfaces. In our previous studies, 7-mer peptides that specifically bind to film surfaces composed of stereoregular isotactic (it) poly(methyl methacrylate) (PMMA),8d syndiotactic (st) PMMA,8e and st-polystyrene8f have already been identified by the PD method. Our observations suggested that these peptides have the potential for recognizing polymer stereoregularity-dependent differences in the polymer surfaces, in the amphiphilic polymer surfaces, and in the presence of inclusion solvents. All of these peptides were characterized by ELISA using phage clones; therefore, it is necessary and significant to further confirm the affinities of short peptides freed from phage bodies and to compare the newly obtained quantitative data against data obtained from ELISA. Since stereoregular PMMAs have a simple chemical structure composed of alkyl main chains and lateral methyl/methyl ester groups as well as a high stereoregularity and since the PMMA structure is stable and obvious in an aqueous phase, the affinity analysis of synthetic 7-mer peptides against stereoregular PMMAs by the suitable methods will generally yield further understanding of these peptide-material interactions. In this study, we quantitatively analyzed the affinity processes of free nonlabeled 7-mer peptides against target it-PMMA and a structurally similar reference st-PMMA based on real-time SPR measurements. We demonstrated extremely high specificity of the short peptides against the target, followed by an estimation of the association (k1)/dissociation (k-1) rate constants and the ratio affinity constants (Ka ) k1/k-1). Our results were comparable with previous ELISA data.8d Essential aa’s were revealed by changing each aa to an inert Ala. The affinities of shorter 4-mer peptides containing essential aa’s were also demonstrated.

Possible conformations of superior peptides were obtained by Molecular Mechanics methods. Thermodynamic parameters were also obtained by affinity analysis at various temperatures, suggesting a peptide affinity mechanism. The 7-mer peptides directed against the target st-PMMA8e as well as against the reference it-PMMA were analyzed similarly as an advanced system. To our knowledge, this is the first study that systematically reports highly specific rate and affinity constants of extremely short material-binding peptides.

(13) Chen, H.; Su, X.; Neoh, K.-G.; Choe, W.-S. Anal. Chem. 2006, 78, 4872. (14) Tamerler, C.; Oren, E. E.; Duman, M.; Venkatasubramanian, E.; Sarikaya, M. Langmuir 2006, 22, 2712.

(15) (a) Hatada, K.; Ute, K.; Okamoto, Y.; Kitayama, T. Polym. J. 1986, 18, 1037. (b) Kitayama, T.; Shinozaki, T.; Sakamoto, T.; Yamamoto, M.; Hatada, K. Makromol. Chem 1989, 15, 167.

Experimental Section Polymer Synthesis. it- and st-PMMAs were synthesized following conventional living anionic polymerization methods.15 In brief, methyl methacrylate (MMA, Wako Chemicals) was distilled and then polymerized in distilled toluene at -78 °C with freshly prepared t-C4H9MgBr15a and t-C4H9Li/(C2H5)3Al15b as initiators for the itand st-PMMAs, respectively. The number-average (Mn) and weightaverage (Mw) molecular weights were measured by size exclusion chromatography (GPC-8020, Tosoh; eluent ) tetrahydrofuran) on a TSKguard column SuperH-H with one column each of TSKgel SuperH2000 and SuperH4000 (Tosoh) at 40 °C at a flow rate of 1.0 mL min-1. Calibration was demonstrated by using commercially available PMMA standards (Polymer Laboratories). The tacticity (mm:mr:rr) was measured by using 1H NMR signals from the R-methyl protons at 1.0-1.5 ppm. it-PMMA (Mn ) 35 500, Mw/Mn ) 1.1, mm:mr:rr ) 98:2:0) and st-PMMA (Mn ) 28 200, Mw/Mn ) 1.3, mm:mr:rr ) 0:11:89) were used for SPR measurements. SPR Measurements. Both Biacore 3000 and X were used for the SPR measurements (the kinetic data were essentially the same within experimental error). Gold-coated glass slides (SIA Kit Au, Biacore) were coated with approximately 10 nm thick films composed of it-PMMA or st-PMMA and set on the SPR apparatus. it-PMMA specific peptides with a free N-terminus and amidated C-terminus were purchased from Sigma Genosys (>95% purity or immunograde) and were purified by high performance liquid chromatography (ELITE LaChrom, Hitachi Hitechnologies) with Cosmosil 5C18AR-300 (Nacalai Tesque) using acetonitrile-water solvents before use. st-PMMA specific peptides were purchased from Invitrogen (95%). The peptides were dissolved in 10 mM HEPES buffer containing 150 mM NaCl (pH 7.4, Biacore) at the appropriate concentrations. The freshly prepared peptide solutions were then applied to the polymer film surface at a flow rate of 20 µL min-1 at 25 °C (for thermodynamic analyses, 15-25 °C) for 2 min (association), and then the peptide solutions were eliminated to the buffer solution under the same conditions for 2 min (dissociation). The resulting sensorgrams at 4-5 concentrations were fitted by global fitting analysis, which simultaneously fits all sensorgrams including association/dissociation processes and the responses due to the rapid changes in the bulk refractive indices when the flow of the peptide solutions (association) and buffer solutions (dissociation) start, using BIAevaluation software version 4.1 (assuming a 1:1 Langmuir binding model), followed by an estimation of k1 (M-1 s-1) and k-1 (s-1). Finally, Ka was estimated by the following equation: Ka ) k1/k-1 (M-1). The baseline drift was maintained at less than 0.3 resonance units (RU) min-1 during the kinetic measurements. The fitting result evaluated by the chi-squared (χ2) value (an index of fitting reliability) was less than 8.7 for all peptides. Although the χ2 value fluctuates based on small background noises of the sensorgrams, values of less than 10 are considered to be acceptable according to the BIAevaluation handbook. Relatively slow dissociation processes were monitored in this study; however, previous SPR measurements revealed similarly ordered rate constants as those analyzed in this study, suggesting that the present method adequately evaluated peptide specificities.16 Even though the dissociation processes were monitored for a much longer time (typically a 30 min dissociation),

Highly Specific Affinities of Short Peptides

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Figure 1. SPR sensorgrams of the c02 peptide against (a) it-PMMA and (b) st-PMMA at various concentrations. the resulting rate constants were almost the same, thereby indicating that monitoring a 2 min dissociation with relatively smaller χ2 values was sufficient to reliably analyze the rate constants. Fluorescence Microscopic Observations. Approximately 40 nm thick PMMA films were coated onto glass slides (Matsunami). Peptide solutions at 1.6 mM were mounted on the films for 30 min at ambient temperatures, and the films were rinsed with TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.5) and ultrapure water (MilliQ). The samples then underwent fluorescence microscopy (Leica, DM6000 B) (excitation wavelength ) 360 nm; emission wavelength ) 470 nm; half-width ) 40 nm) in air.

Results and Discussion Observation of Peptide Affinities. The time-dependent affinities of short peptides against the polymer film surfaces were successfully detected by highly sensitive SPR measurements. Figure 1 shows typical examples of SPR sensorgrams composed of the association and dissociation processes for a c02 peptide (Glu-Leu-Trp-Arg-Pro-Thr-Arg), which was previously identified as an it-PMMA specific peptide,8d against it- and st-PMMAs. The initial drastic increases in resonance units (RU) can be attributed to changes in the refractive indices of the bulk solutions, and they are not derived from the peptide association. Next, the gradual RU change, which was quantitatively converted to peptide association, could be observed. The subsequent substitution of the c02 solution to the buffer using the SPR flow system resulted in drastic RU decreases, which were also due to refractive index changes. After that, adequate amounts of the peptide remained, and the bound peptides dissociated with time. The sensorgrams (16) (a) Haseley, S. R.; Talaga, P.; Kamerling, J. P.; Vliegenthart, J. F. G. Anal. Biochem. 1999, 274, 203. (b) Chiou, S.-T.; Chen, Y.-W.; Chen, S.-C.; Chao, C.-F.; Liu, T.-Y. J. Biol. Chem. 2000, 275, 1630. (c) Jenkins, J. L.; Lee, M. K.; Valaitis, A. P.; Curtiss, A.; Dean, D. H. J. Biol. Chem. 2000, 275, 14423.

Figure 2. Fluorescence microscopic analyses of surfaces coated and not coated (control) with the c02 peptide. (a) and (b) show fluorescence images of peptide-coated it-PMMA surfaces after 0 and 50 s, respectively. (c) shows the time-dependent photobleaching of it-PMMA surfaces coated and not coated (control) with the c02 peptide in the areas squared in (a) and (b). (d-f) are the same experiments for st-PMMA surfaces. The white bars in (a), (b), (d), and (e) show 50 µm scale. The overlap of the peptide photobleaching in (c) suggests that the c02 peptide homogeneously covered the it-PMMA surface at various selected areas.

were suitably fitted to obtain the kinetic parameters. It was obvious from the sensorgrams that greater amounts of the c02 peptide were bound to the target it-PMMA as compared to the reference st-PMMA at similar concentrations, thus suggesting the specificity of the c02 peptide for it-PMMA. Fluorescence microscopic observations of the it- and st-PMMA film surfaces after c02 binding are shown in Figure 2. The fluorescence derived from the excited Trp residues of the c02 peptide clearly bleached with time. Considering that the photobleaching is correlated to the bound c02 peptide, these observations suggest that greater amounts of the c02 peptide are bound onto the it-PMMA surfaces. The aforementioned observation coincided with that from the SPR measurements, as shown in Figure 1. Furthermore, the bleaching intensity was approximately the same at various areas, thereby indicating that peptides bound to the polymer surfaces homogeneously over micrometer-sized areas. It is noted that the bleaching for bare PMMA surfaces could be ignored. Quantification of Peptide Affinities. Previously isolated peptides directed against it-PMMA underwent SPR measurements. The kinetic parameters and Ka of six peptides with different sequences as well as the apparent association constants (Kapp) of

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Table 1. SPR Kinetic Parameters for Peptides against PMMA peptide

sequence

k1 (M-1 s-1)

k-1 (10-3 s-1)

Ka (103 M-1)

c02 c18 c04 c26 c03 c10d

ELWRPTR ARPHLSF SSPWMRE GIRHTNR QLQKYPS NLQEFLF

31 (5.5)c 13 (2.0) 15 (1.1) 20 (1.6) 13 (0.22) nde (nd)

0.11 (0.80) 0.20 (1.3) 0.24 (0.25) 0.48 (1.5) 0.58 (0.19) nd (nd)

280 (6.9) 65 (1.5) 62 (4.2) 42 (1.1) 23 (1.2) E1A > R4A. Our previous ELISA study using phage clones concluded that the Arg-Pro-Thr-Arg motif of the c02 peptide, which is composed of aa’s with proton-donor hydroxy and amino lateral groups adjacent to the Pro, was an essential motif for affinity strength and specificity.8d The aforementioned observations from Ala scanning directly verified the importance of the sequence using free peptides. A rigid conformation of the sequence, which would be produced by a kinked Pro, must three-dimensionally arrange the lateral hydroxyl and amino groups of the Thr and two Arg residues as proton-donors to successfully recognize the position of the it-PMMA ester groups through hydrogen bonding. Glu, in which the Ala substitution for the E1A mutant decreased the Ka value moderately, might participate in additional hydrogen bonding interactions. To stably form the regular structure of the c02 peptide, a great conformational change of the c02 peptide must be necessary; the induced fit mechanism, which sufficiently explains the peptide conformational change, is proposed by the analysis of the thermodynamic parameters (see below). As a consequence, Ala scanning revealed the absolutely essential aa’s for it-PMMA recognition. The formation of hydrogen bonding interactions between it-PMMA and the c02 peptide was analyzed by attenuated total reflection infrared spectroscopy using the refractive surface of 100 nm thick gold-coated poly(ethylene terephthalate) substrates (Tanaka Precious Metals, Japan) with a Perkin-Elmer Spectrum One (Perkin-Elmer) instrument in air; however, that was not successful due to detection limitations. To confirm the potential of the it-PMMA recognizing motif, the affinities of the C-terminal Arg-Pro-Thr-Arg and the N-terminal Glu-Leu-Trp-Arg of the c02 peptide against it- and st-PMMAs were analyzed as shown in Table 3. The Ka value for the former peptide was determined to be 4.6 × 104 M-1 against it-PMMA. The value was smaller than that for the original c02 peptide, which was mainly due to the decrease in k1. However, the value was significantly greater than that for the c02 peptide against the reference st-PMMA. Furthermore, the Ka ratio between it-PMMA and st-PMMA was almost the same as that of the original peptide, indicating that the shorter peptide composed of only four aa residues still showed great specificity. On the other hand, the Ka value for the latter peptide was 1.6 × 103 M-1

Table 3. SPR Kinetic Parameters for 4-mer Peptides against PMMA peptide c02 4-7 1-4 b

sequence

k1 (M-1 s-1)

k-1 (10-3 s-1)

Ka (103 M-1) Ka,it/Ka,sta

ELWRPTR 31 (5.5)b 0.11 (0.80) 280 (6.8) RPTR 7.4 (0.59) 0.16 (0.63) 46 (0.94) ELWR 2.5 (1.5) 3.0 (0.57) 1.6 (5.2)

41 49 0.31

a Ratios between affinity constants against it-PMMA and st-PMMA. Against st-PMMA.

Figure 3. Possible conformations of the Arg-Pro-Thr-Arg peptide (upper) and the c02 peptide (bottom) obtained by Molecular Mechanics methods. N and C show the N- and C-termini, respectively, of the peptides. The peptide main chain, the Pro ring, the Arg guanidium group, and the Thr hydroxyl group are shown in yellow, brown, red, and blue, respectively.

against it-PMMA. This value was much smaller than that for the original c02 peptide, which was due to the great decreases and increases in k1 and k-1, respectively. In addition, the Ka ratio did not show any it-PMMA specificities. The aforementioned observations clearly showed that the C-terminal Arg-Pro-ThrArg of the c02 peptide was an essential motif for it-PMMA recognition and that peptides composed of four aa residues still have the potential to recognize polymer stereoregularities. For peptidyl nanomaterials, shorter peptides would be helpful. The minimization of it-PMMA-recognizing peptides and the maximization of the affinities of tandem or clustered peptides using minimized motifs will be performed in the near future. Possible structures with stable and sterically acceptable conformations for the Arg-Pro-Thr-Arg peptide and the c02 peptide in a vacuum were obtained by energy optimization from extended chain conformations using Molecular Mechanics (Insight II version 98, Accelrys Software Inc.), which considers the sum of the nonbond interaction energy such as van der Waals attraction, van der Waals repulsion, and electrostatic interactions, as shown in Figure 3. The Arg-Pro-Thr-Arg peptide potentially forms a hairpin structure with Pro hinges (due to steric hindrance, PMMA chains cannot be inserted inside the hairpins). Threedimensionally organized lateral groups of Arg and Thr in the rigid conformation might interact effectively with the it-PMMA. Interestingly, the Arg-Pro-Thr-Arg sequence of the c02 peptide showed a similar conformation to the Arg-Pro-Thr-Arg peptide, thus supporting the idea that the specificity of the c02 peptide is determined by the Arg-Pro-Thr-Arg sequence. Note that the Ka ratios were also the same between the Arg-Pro-Thr-Arg peptide and the c02 peptide. The greater Ka value of the c02 peptide than that of the Arg-Pro-Thr-Arg peptide against it-PMMA seemed to be caused by additional interactions from the N-terminal Glu-

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Serizawa et al. Table 4. Thermodynamic Parameters for the c02 Peptide

Figure 4. The dependence of the ln Ka value of the c02 peptide against T-1 for the (a) it-PMMA and (b) st-PMMA surfaces. The data were fitted to a first-order linear relationship, assuming the van’t Hoff equation.

Leu-Trp (especially the hydrogen bonds of Glu). Note that the mutation of Glu to Ala (E1A) moderately decreased the Ka value against it-PMMA. Thermodynamic Analysis of Peptide Affinities. The aforementioned Ala scanning of the c02 peptide and the affinity analyses of the shorter 4-mer peptides suggested that a regularly formed conformation of the c02 peptide is essential for its itPMMA affinities. To further understand these peptide affinity mechanisms, the thermodynamic parameters for the c02 peptide against the it- and st-PMMA surfaces were estimated by analyzing the Ka value at various temperatures. Figure 4 shows the dependence of the ln Ka value against T-1. The Ka value decreased with increasing temperature, suggesting that the predominant driving force for the peptide affinities against PMMAs was derived from hydrogen bonding interactions. The data plots were fitted to a first-order linear relationship, assuming the van’t Hoff equation. The enthalpy change (∆H°) and the entropy change (∆S°), which were estimated from the slope and the Y-inset, respectively, as well as the Gibbs free energy change (∆G°) for the c02 peptide are shown in Table 4. The -∆H° value for the target it-PMMA was greater than that for the reference st-PMMA, suggesting that the peptide side chains interacted with the target ester groups through more multiple hydrogen bonding interactions as compared with the reference. Interestingly, the -∆S° value for the target was greater than that for the reference, possibly

polymer

∆G° (kJ mol-1)

∆H° (kJ mol-1)

∆S° (J mol-1 K-1)

it-PMMA st-PMMA

-31 -22

-35 -23

-12 -5

suggesting that greater conformational changes of the peptide and/or it-PMMA occurred to achieve the specific affinity. This entropic disadvantage was compensated for by the aforementioned enthalpy gain. Therefore, it may be acceptable to consider that the specific affinity occurred through an induced fit process. The induced fit processes of material-binding short peptides have already been proposed by molecular dynamics simulations of circular 7-mer peptides directed against platinum surfaces.19 As a consequence, thermodynamic analysis of the peptide affinities revealed some mechanistic insight into the specific peptide affinity against it-PMMA. It is noted that this is the first study to experimentally demonstrate the thermodynamic parameters of material-binding peptides. Peptide Affinities for st-PMMA. Our previous studies isolated 7-mer peptides that recognized st-PMMA film surfaces8e as well as it-PMMA.8d To understand whether isolated peptides for stPMMA similarly show specific affinities, a typical 7-mer peptide composed of the His-Lys-Pro-Asp-Ala-Asn-Arg sequence was applied to the SPR measurements (note that st-PMMA films were sufficiently conditioned in the buffer solution before the measurements). The resulting k1, k-1, and Ka values for st-PMMA (it-PMMA) were 14 (3.6) M-1 s-1, 0.14 × 10-3 (1.2 × 10-3) s-1, and 9.1 × 104 (3.0 × 103) M-1, respectively. The Ka ratio for it- and st-PMMA was 31, which was also greater than that obtained from ELISA using phage clones. These observations strongly indicate that polymer-binding peptides isolated by the PD method generally show highly specific affinities. In fact, the difference in the static contact angles of water droplets was less than several degrees between the it- and st-PMMA surfaces, indicating that the macroscopic hydrophobicities are similar between the two surfaces.8e Therefore, these peptides do not recognize a difference in simple hydrophobicity, and they must recognize the relative positions of the PMMA ester groups, which are determined by the PMMA stereoregularities.18

Conclusions The association/dissociation processes of synthetic and short 7-mer peptides freed from phage bodies directed against stereoregular PMMA film surfaces were quantitatively detected by SPR measurements. Kinetic parameters such as association/ dissociation constants and affinity constants of the peptides were estimated. The peptides rapidly bound to the target it-PMMA but slowly dissociated from the target as compared to the reference st-PMMA. The affinity profiles were dependent on the peptide sequences. The affinity constants of the peptides for the target ranged from 2.3 × 104 to 2.8 × 105 M-1, which were 15-43fold greater than that for the reference, indicating high peptide specificities. The c02 peptide with the Glu-Leu-Trp-Arg-ProThr-Arg sequence showed the greatest affinity constant and the highest specificity. Assays using phage-displayed peptides were representative of the relative affinity ordering analyzed using free peptides. However, the phage assays underestimated the specificity. Ala scanning of the c02 peptide revealed essential aa’s such as Glu, two Arg, Pro, and Thr for the specific affinities. The substitution of Pro to Ala considerably decreased the affinity constant, suggesting that the rigid and kinked structure formed (19) (a) Oren, E. E.; Tamerler, C.; Sarikaya, M. Nano Lett. 2005, 5, 415. (b) Kantarci, N.; Tamerler, C.; Sarikaya, M.; Haliloglu, T.; Doruker, P. Polymer 2005, 46, 4307.

Highly Specific Affinities of Short Peptides

by Pro is the most important for peptide affinity. The shorter 4-mer peptide (Arg-Pro-Thr-Arg) included in the c02 C-terminus, which contained four essential aa’s, still showed specific affinities, although the N-terminal 4-mer peptide (Glu-Leu-Trp-Arg) did not show any specificity. Possible hairpin conformations of the essential 4-mer sequence were obtained by Molecular Mechanics methods. Decreases in the affinity constants at higher temperatures suggested that hydrogen bonding interactions were predominantly responsible for the c02 peptide affinities. The thermodynamic parameters of the c02 peptide were significantly different between the target and the reference. A greater enthalpy gain, which compensates for a greater entropy loss, suggested an induced fit mechanism for the target affinities. The high peptide specificities were generally confirmed by 7-mer peptides that recognized the target st-PMMA. The appropriate utilization of short peptides

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for affinity assays promises to give essential information about the material recognition characteristics of these peptides. The present study clearly demonstrated that extremely short peptides can potentially recognize slight differences in organic materials with simple chemical structures. Acknowledgment. Authors thank Dr. K. Matsumura (Shibaura Institute of Technology) for helpful discussions, Dr. Y. Yong, Mr. T. Morishita, and Mr. T. Chiaki (Leica) for help with fluorescence microscopic observations, and Prof. K. Kudo and Dr. S. Sakamoto (The University of Tokyo) for help with Molecular Mechanics. LA701822N