Evaluation of Interaction Forces between Profilin ... - ACS Publications

Mar 12, 2008 - The single molecular retraction force between peptide probe 1 and profilin was estimated to be 96 pN, as determined by Gaussian fitting...
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Langmuir 2008, 24, 4050-4055

Evaluation of Interaction Forces between Profilin and Designed Peptide Probes by Atomic Force Microscopy Tomoko Okada, Masato Sano, Yuji Yamamoto, and Hiroshi Muramatsu* School of Bionics, Tokyo UniVersity of Technology, Katakura, Hachioji, Tokyo 192-0982, Japan ReceiVed October 26, 2007. In Final Form: February 4, 2008 We evaluated the binding affinity of peptide probes for profilin (protein) using force curve measurement techniques and atomic force microscopy (AFM). The peptide probes designed and synthesized for this investigation were H-A3GP5GP5GP5G-OH (1), H-A3GP5G-OH (2), H-A3G7-OH (3), and H-A3G-OH (4). Each peptide probe was immobilized on a cantilever tip, and the interaction force to profilin, immobilized on a mica substrate, was examined by force curve measurements. The retraction forces obtained showed a sequence-dependent affinity of the peptide probe for profilin. The retraction force for peptide probe 1 was the largest of the four probes examined, and it confirmed that peptide probe 1 has high affinity for profilin. The single molecular retraction force between peptide probe 1 and profilin was estimated to be 96 pN, as determined by Gaussian fitting to the histogram of the retraction forces.

Introduction Proteins display a wide variety of biological activities. Elucidation of the interaction of proteins at the atomic level can lead to advancements in areas such as those concerned with curative diseases and the development of new medicines. A variety of methods have been reported for the evaluation of proteinprotein interactions including the use of electrophoresis, twohybrid methods, the enzyme-linked immunosorbent assay (ELISA), and the fluorescence resonance energy transfer (FRET) method. These methods represent versatile techniques that have been widely used in the evaluation of protein affinity.1,2 Atomic force microscopy (AFM) has been utilized as an advanced method for the evaluation of affinity under in Vitro conditions. By measuring a force curve, an adhesive force between a substrate and a tip on a cantilever can be detected as the bending amplitude of the cantilever in a separating motion of the tip from the substrate after approach and contact has been made. Many researchers have examined force curves and succeeded in evaluating interactions between one biomolecule immobilized on a cantilever tip and another biomolecule on the substrate, with examples including DNA-DNA, protein-protein, and biotin-avidin interactions.3-13 In recent years, the force curve measurement * To whom correspondence [email protected].

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(1) Fields, S.; Song, O. Nature 1989, 340, 245-246. (2) Sekar, R. B.; Periasamy, A. J. Cell Biol. 2003, 160, 629-633. (3) Lee, G. U.; Chrisey, L. A.; Colton, R. J. Science 1994, 266, 771-773. (4) Blank, K.; Mai, T.; Gilbert, I.; Schiffmann, S.; Rankl, J.; Zivin, R.; Tackney, C.; Nicolaus, T.; Spinnler, K.; Oesterhelt, F.; Benoit, M.; Clausen-Schaumann, H.; Gaub, H. E. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 11356-11360. (5) Rief, M.; Gautel, M.; Oesterhelt, F.; Fernandez, J. M.; Gaub, H. E. Science 1997, 276, 1109-1112. (6) Weisel, J. W.; Shuman, H.; Litvinov, R. I. Curr. Opin. Struct. Biol. 2003, 13, 227-235. (7) Scha¨fer, C.; Eckel, R.; Ros, R.; Mattay, J.; Anselmetti D. J. Am. Chem. Soc. 2007, 129, 1488-1489. (8) Takeda, S.; Ptak, A.; Nakamura, C.; Miyake, J.; Kageshima, M.; Jarvis, S. P.; Tokumoto, H. Chem. Pharm. Bull. 2001, 49, 1512-1516. (9) Sekiguchi, H.; Ikai, A.; Arakawa, H.; Sugiyama, S. e-J. Surf. Sci. Nanotechnol. 2006, 4, 149-154. (10) Han, S. P.; Yoda, S.; Kwak, K. J.; Suga, K.; Fujihira, M. Ultramicroscopy 2005, 105, 148-154. (11) Eckel, R.; Wilking, S. D.; Becker, A.; Sewald, N.; Ros, R.; Anselmetti, D. Angew. Chem., Int. Ed. 2005, 44, 3921-3924. (12) Eckel, R.; Ros, R.; Decker, B.; Mattay, J.; Anselmetti, D. Angew. Chem., Int. Ed. 2005, 44, 484-488. (13) Bartels, F. W.; Mclntoshi, M.; Fuhrmann, A.; Metzendorf, C.; Plattner, P.; Sewald, N.; Anselmetti, D.; Ros, R.; Becker, A. Biophys. J. 2007, 92, 43914400.

has been applied to the evaluation of the affinity of proteins on a living cell surface.14-17 These force measurement techniques can provide information pertaining to the force of the interaction present in addition to an exploration of the energy landscape of the interaction between biomolecular pairs. For the evaluation of protein affinity, a protein probe should be immobilized to the cantilever tip. This task remains problematic, since protein probes are not always easy to obtain, and orientation of a protein on the cantilever tip so as to retain an accessible interacting region may prove difficult. In contrast, peptide probes possess simpler structures compared with protein probes, and the sequence of peptide probes can easily be varied using uncomplicated synthetic procedures such as those employing an automated synthesizer. Consequently, the use of peptide probes offers advantages for the systematic investigation of sequence-dependent affinity interactions. Although the application of peptide probes has already been reported in the analysis of protein-protein interactions using electrophoresis, surface plasmon resonance, and quartz crystal microbalance, only a few reports have dealt with the use of force curve measurements.8,14 In this study, we applied peptide probes for the evaluation of protein affinity by measuring force curves. Using cantilever tips modified with various peptide probes allows for the investigation of sequencedependent interactions of proteins immobilized on the substrate. Profilin was used as a model protein, since it plays an interesting role in regulating the polymerization of actin filaments in ViVo. Profilin displays high affinity for vasodilator-stimulated phosphoprotein (VASP), where the binding region of VASP contains a proline-rich segment. Poly-L-proline interacts with profilin in a manner that may involve two binding modes as revealed by Mahoney et al.18,19 Given the importance of proline-rich segments in relation to profilin binding, we designed peptide probes and examined their respective affinities for profilin. (14) Lehenkari, P. P.; Horton, M. A. Biochem. Biophys. Res. Commun. 1999, 259, 645-650. (15) Uehara, H.; Osada, T.; Ikai, A. Ultramicroscopy 2004, 100, 197-201. (16) Grandbois, M.; Dettmann, W.; Benoit, M.; Gaub, H. E. J. Histochem. Cytochem. 2000, 48, 719-724. (17) Yoshino, T.; Sotome, I.; Ohtani, T.; Isobe, S.; Oshita, S.; Maekawa, T. J. Electron Microsc. 2000, 49, 483-486. (18) Mahoney, N. M.; Rozwarski, D. A.; Fedorov, E.; Fedorov, A.; Almo, S. C. Nature Struct. Biol. 1999, 6, 666-671. (19) Mahoney, N. M.; Jammy, P. A.; Almo, S. C. Nature Struct. Biol. 1997, 4, 953-960.

10.1021/la703344u CCC: $40.75 © 2008 American Chemical Society Published on Web 03/12/2008

Binding Affinity of Peptide Probes for Profilin

Experimental Section Reagents. Fmoc-protected amino acids Fmoc-L-Ala-OH, FmocL-Pro-OH, Fmoc-Gly-OH, and Fmoc-Gly-Wang resin were purchased from Kokusan Chemicals. N-Methylmorpholine, trifluoroacetic acid (TFA), and 3-aminopropyltriethyoxysilane (APTES) were purchased from Tokyo Kasei Kogyo, glutaraldehyde (GA) was from Wako, and dithiobis(succinimidyl undecanoate) (DSU) was from Dojindo. Profilin was purchased from Funakoshi. The profilin purchased includes two isoforms (profilin I and IIa). Phosphate buffer was prepared as a 10 mmol/L solution and adjusted to pH 7.4. All reagents were used without further purification, and reactions were performed at room temperature unless otherwise indicated. Synthesis of Peptide Probes. Peptide probes H-A3GP5GP5GP5GOH (1), H-A3GP5G-OH (2), H-A3G7-OH (3), and H-A3G-OH (4) were synthesized by the solid-phase method using Fmoc chemistry carried out with a PSSM-8 peptide synthesizer (Shimadzu). Synthesized peptides were cleaved from the resin using TFA, and crude products were purified by reversed phase column chromatography (RP-HPLC) using LC-8A and SPD-M10A (Shimadzu). Analytical column chromatography was performed using a CAPCELL PAK C18 MGII column (1.0 mm × 250 mm; Shiseido), and titled compounds were collected using a CAPCELL PAK C18 MGII column (4.6 mm × 250 mm; Shiseido). Purified products were identified by electrospray ionization time-of-flight mass spectrometry using a JMS-T100LC spectrometer (JEOL), and 1H NMR spectra were recorded using a 400 MHz FT-NMR spectrometer (Bruker). The analytical results of the peptide probes are shown below. Peptide Probe 1. Yield: 60%. RP-HPLC (acetonitrile/H2O ) 1:5 (0.1% TFA), 1.0 mL/min): Rt ) 3.7 min. MS: m/z (M ) C90H134N22O23) ) 1937.9 [M + Na]+ (calcd: 1938.0), 1916.0 [M + H]+ (calcd: 1916.0), 969.5 [M + H + Na]2+ (calcd: 969.5), 958.5 [M + 2H]2+ (calcd: 958.5), 639.3 [M + 3H]3+ (calcd: 639.3). Peptide Probe 2. Yield: 75%. RP-HPLC (acetonitrile/H2O ) 1:5 (0.1% TFA), 1.0 mL/min): Rt ) 4.1 min. MS: m/z (M ) C38H58N10O11) ) 869.4 [M + K]+ (calcd: 869.4), 853.4 [M + Na]+ (calcd: 853.4), 831.4 [M + H]+ (calcd: 831.4), 427.2 [M + H + Na]2+ (calcd: 427.2), 416.2 [M + 2H]2+ (calcd: 416.2). 1H NMR (CD3OD, 400 MHz): δ ) 4.72-4.65 (m, 3H; CH(Pro)), 4.52 (br, 1H; CH(Pro)), 4.46 (dd, J ) 3.6, 8.4 Hz, 1H; CH(Pro)), 4.40 (q, J ) 5.2 Hz, 2H; CH(Ala)), 4.15 (d, J ) 17.2 Hz, 1H; CHH(Gly)), 4.00 (d, J ) 18.0 Hz, 1H; CHH(Gly)), 3.91 (d, J ) 16.8 Hz, 1H; CHH(Gly)), 3.90 (q, J ) 6.8 Hz, 1H; CH(Ala)), 3.87-3.54 (m, 11H; CH(Gly), CH2(Pro)), 2.36-1.94 (m, 20H; CH2(Pro)), 1.51 (d, J ) 6.8 Hz, 3H; CH3(Ala)), 1.40 (d, J ) 7.6 Hz, 3H; CH3(Ala)), 1.38 (d, J ) 7.6 Hz, 3H; CH3(Ala)). Peptide Probe 3. Yield: 78%. RP-HPLC (acetonitrile/H2O ) 1:5 (0.1% TFA), 1.0 mL/min): Rt ) 2.9 min. MS: m/z (M ) C23H38N10O11) ) 669.3 [M + K]+ (calcd: 669.2), 653.3 [M + Na]+ (calcd: 653.3), 631.3 [M + H]+ (calcd: 631.3). Peptide Probe 4. Yield: 82%. RP-HPLC (acetonitrile/H2O ) 1:5 (0.1% TFA), 1.0 mL/min): Rt ) 3.1 min. MS: m/z (M ) C11H20N4O5) ) 311.1 [M + Na]+ (calcd: 311.1), 289.1 [M + H]+ (calcd: 289.2). 1H NMR (CD OD, 400 MHz): δ ) 4.39 (q, J ) 7.2 Hz, 2H; CH(Ala)), 3 3.93 (q, J ) 7.2 Hz, 1H; CH(Ala)), 3.97 (d, J ) 18.0 Hz, 1H; CH(Gly)), 3.86 (d, J ) 17.6 Hz, 1H; CH(Gly)), 1.52 (d, J ) 6.8 Hz, 3H; CH3(Ala)), 1.40 (q, J ) 7.2 Hz, 3H; CH3(Ala)), 1.38 (d, J ) 7.2 Hz, 3H; CH3(Ala)). Immobilization of Peptide Probes on Cantilever Tips. Peptide probes 1-4 were covalently immobilized on cantilever tips. The silicon nitride cantilever (Olympus, OMCL-TR400PB-1) covered with Au was immersed in 1 M HCl solution for 5 min followed by rinsing with H2O. The cantilever was treated with DSU dissolved in acetonitrile (1 mmol/L) and then kept in the solution for 12 h to facilitate covering of the surface with a self-assembled monolayer (SAM). After rinsing with acetonitrile, the cantilever was treated with 1 mmol/L peptide probe dissolved in acetonitrile/H2O ) 1:1 for 12 h. Following the peptide probe immobilization procedure, the cantilever was treated with Tris-HCl buffer (10 mmol/L, pH 8) for 12 h to block the remaining succinimidyl groups on the SAM. The cantilever was subsequently rinsed with H2O and then kept in a desiccator.

Langmuir, Vol. 24, No. 8, 2008 4051 X-ray Photoelectron Spectroscopy (XPS). XPS was performed using a JPS-9200 spectrometer (JEOL) to confirm that immobilization of the peptide probe had occurred. Spectra were acquired using unmonochromatized Mg KR radiation (photon energy ) 1253.6 eV) at a 50 eV pass energy and averaged for 20 scans. Peak positions were assigned by referencing the C 1s peak to a binding energy of 285 eV. Substrates for the XPS measurements were prepared on a thin Au film coated on a polished quartz face. The gold surface was treated with piranha solution (H2SO4/H2O2 ) 70:30 (v/v)) for 15 min, rinsed with water, and then dried under vacuum.20 The cleaned substrate was treated with 1 mmol/L DSU dissolved in absolute acetonitrile for 12 h. After rinsing with acetonitrile, the substrates were dried under vacuum. To immobilize peptide probes, the dried substrate with a SAM was immersed in a solution of peptide probe 1 (1 mmol/L in acetonitrile/H2O ) 1:1) for 12 h. The substrate was washed with acetonitrile/H2O ) 1:1 followed by H2O and then dried under vacuum. Fourier Transform Infrared Spectroscopy (FT-IR). FT-IR was performed using a FTS3000/600UMA spectrometer (Varian) equipped with a grazing angle objective to confirm the immobilization reaction of the peptide probes. Interferograms were collected at 4 cm-1 resolution at an angle of incidence of 65-85°, and the spectra obtained were averaged for 1024 scans. Substrates were prepared using the same procedure as described in the XPS measurement section. For both the XPS and FT-IR measurements, substrates were used without quenching the activated carboxylate groups by Tris-HCl buffer. Immobilization of Profilin on Mica Substrate. Profilin was covalently immobilized on a mica surface using a previously reported general procedure.21 A slip of mica was glued on a glass slide using UV adhesive and irradiation with UV light at 365 nm for 10 min. The surface layer of the mica was stripped to obtain a clean surface of silanol groups, and the freshly prepared mica surface was treated with APTES (vapor) for 1 h. The surface was rinsed with distilled water and dried in vacuum. The dried mica surface was covered with 0.1% GA aqueous solution, kept in this solution for 1 h, and then rinsed with H2O. The activated mica surface was then treated with profilin solution (0.1 mg/mL in phosphate buffer) for 1 h to immobilize profilin. After rinsing with phosphate buffer, the mica surface was treated with Tris-HCl (10 mmol/L, pH 8) for 12 h to block any active aldehyde groups. The prepared mica substrate was kept in phosphate buffer to prevent drying. A topographic image of the profilin-immobilized mica surface obtained by AFM is included in the Supporting Information. Force Curve Measurements. Force curves were measured using an atomic force microscope (SPI4000, Seiko Instruments). The spring constant of the cantilever was calibrated using the thermal vibration method.22 The mica substrate with immobilized profilin was placed in a fluid cell containing phosphate buffer (10 mmol/L, pH 7.4) for a control measurement or phosphate buffer with free peptide probe 1 (1 mmol/L) for a blocking experiment. The force curve measurement was carried out in the fluid cell using the cantilever tip modified with the peptide probes, and force curves were recorded at a velocity of 120 nm s-1. Every force curve was measured at different points on the substrate, and each point was manually set within a squared area of ∼200 nm in width. Several hundred force curves were recorded at different positions on the substrate, and retraction forces were combined from the recorded force curves (670 curves for peptide probe 1, 300 curves for peptide probe 2, 300 curves for peptide probe 3, and 120 curves for peptide probe 4). Retraction forces are summarized in the form of a histogram and represented as a probability (%) that was obtained by dividing each count number by the total number of measured force curves.

Results and Discussion For the purpose of evaluating specific interaction forces, four peptide probes having different amino acid sequences were (20) Lo, Y.-S.; Huefner, N. D.; Chan, W. S.; Dryden, P.; Hagenhoff, B.; Beebe, T. P., Jr. Langmuir 1999, 15, 6522-6526. (21) Wang, H.; Bash, R.; Yodh, J. G.; Hager, G. L.; Lohr, D.; Lindsay, S. M. Biophys. J. 2002, 83, 3619-3625. (22) Hutter, J. L.; Bechhoefer, J. ReV. Sci. Instrum. 1993, 64, 1868-1873.

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Figure 2. FT-IR spectra of the gold substrates modified with (a) a SAM and (b) peptide probe 1 immobilized to the SAM. The spectrum of the unmodified gold substrate was used as the reference for these spectra.

Figure 1. Designed peptide probes (a) H-A3GP5GP5GP5G-OH (1), (b) H-A3GP5G-OH (2), (c) H-A3G7-OH (3), and (d) H-A3G-OH (4).

designed. The chemical structures of the peptide probes are shown in Figure 1. Peptide probe 1 (H-A3GP5GP5GP5G-OH) includes three repetitions of GP5 that correspond to the binding region of VASP to profilin.23,24 The GP5GP5GP5 moiety is linked to three consecutive alanine residues (A3) that enables flexible movement of the peptide probe on a substrate. Peptide probe 2 (H-A3GP5G-OH) possesses only one GP5 moiety linked to the trialanine linker. Peptide probes 3 (H-A3G7-OH) and 4 (H-A3G-OH) do not include the GP5 moiety but possess the same trialanine linker that is contained within peptide probes 1 and 2. The four peptide probes 1-4 were synthesized by typical solid-phase syntheses and subsequently purified by HPLC. Peptide probes 1-4 were immobilized on the cantilever tip using the chemical reactions as shown in Scheme 1. In the first step, the cantilever tip was treated with DSU that possesses alkanethiol groups and activated carboxylate groups. The alkanethiol group anchors DSU on the Au surface to form a SAM, and the activated carboxylate groups cover the surface. The SAM on the gold surface is expected to reduce nonspecific interactions between the bare gold surface and protein.25 In the second step, the activated carboxylate groups reacted with the N-terminal end of the peptide probe. In the third step, any remaining activated carboxylate groups were blocked using TrisHCl buffer. The amine groups of Tris react with residual activated carboxylate groups and completely inactivate them. This

inactivation process is important to prevent the tip surface from binding unwanted molecules. Immobilization of the peptide probe was confirmed by FT-IR (Figure 2) and XPS (Figure 3). The cantilever tip surface is so small (on the nanometer scale) that direct analysis of its surface requires the use of specialized equipment with high sensitivity such as a time-of-flight secondary-ion mass spectroscopy (TOFSIMS).20 In this study, we used an alternative substrate having a larger surface area than the cantilever tip. The results of the analyses on the substrate are equivalent to the chemical reaction on the cantilever tip, since both the cantilever tip and the alternative substrate have a Au surface. The FT-IR spectrum of the gold surface with DSU (Figure 2a) showed absorbance at around 1750 cm-1 derived from the carbonyl groups of the succinimidyl moiety. Following immobilization of peptide probe 1, the absorbance at around 1750 cm-1 disappeared, and two peaks at around 1650 and 1550 cm-1 appeared. These two peaks are ascribed to the amide I and amide II groups that are characteristic of peptide chains.25,26 The disappearance of absorbance at around 1750 cm-1 and the appearance at around 1650 and 1550 cm-1 suggests that peptide probe 1 reacted with the succinimidyl group on the gold substrate. The absorbance region for alkyl groups (∼2800-3100 cm-1) was also examined in an effort to obtain further structural information; however, no obvious absorbance was observed (see full range spectra in the Supporting Information). The absence of absorbance at around 2800-3100 cm-1 may have to do with limitations of sensitivity. Furthermore, XPS was utilized to confirm the immobilization of peptide probe 1. The XP spectra for both substrates modified with DSU (Figure 3b) and peptide probe 1 on DSU (Figure 3c) showed clear signals at the binding energy ascribed to S 2p. These signals at the S 2p region suggest that DSU formed a SAM on the substrate and that the SAM remained on the substrate following immobilization

Scheme 1. Immobilization Procedure of the Peptide Probe on the Cantilever Tip

Binding Affinity of Peptide Probes for Profilin

Langmuir, Vol. 24, No. 8, 2008 4053

Figure 4. Representative force curve observed between peptide probe 1 and (a) profilin and (b) Tris. The gray line represents the force for the approaching step, and the black line represents the force for the retracting step. Figure 3. XP spectra of S 2p and N 1s for (a) the unmodified gold substrate, (b) the gold substrate modified with a SAM, and (c) the gold substrate modified with peptide probe 1 mediated by the SAM.

of the peptide probe. Additionally, both substrates exhibited signals at the N 1s region, where the intensity for DSU is weaker than that of the peptide probe. The larger intensity of N 1s for the peptide probe is reasonable, since each molecule of peptide probe 1 possesses 22 nitrogen atoms, while there are only two nitrogen atoms in each molecule of DSU. Therefore, both FT-IR and XPS results provide evidence for the successful immobilization of peptide probe 1 on the cantilever tip. Interaction forces between each peptide probe 1-4 and profilin were examined using force curve measurements. Figure 4a shows a typical force curve obtained between profilin and peptide probe 1. In the retracting course, the force curve shows several peaks of negative force. The negative force indicates an interaction involving attraction. In contrast, the force curve between Tris and peptide probe 1 showed no peak (Figure 4b). These results suggest that the retraction forces demonstrated by Figure 4a resulted from specific affinity involving peptide probe 1 and profilin. In an effort to estimate the strength of this specific affinity, the maximum force was determined for each force curve of peptide probe 1 against profilin, and these forces are summarized in the form of a histogram as shown in the Supporting Information. The histogram fits well to a Gaussian curve, and the peak position is at 652 pN (average ) 633 pN). In contrast, the average maximum retraction force of peptide probe 1 against Tris is 120 pN. The difference in the average values confirms that peptide probe 1 has specific affinity for profilin. In an effort to investigate the dependency of affinity on peptide sequence, force curves for peptide probes 2, 3, and 4 were examined in the same manner as with peptide probe 1. Additionally, a control experiment was performed by adding free peptide probe 1 to the fluid cell of the profilin-immobilized substrate. Histograms of the maximum retraction forces are summarized in the Supporting Information, and Table 1 shows the averaged maximum retraction forces. In the experiment comprising the addition of free peptide probe, although peptide probe 1 showed an averaged retraction force to blocked profilin

Table 1. Averaged Retraction Force Observed between Each Peptide Probe and Profilin (or Blocked Profilin) cantilever

substrate

retraction force (pN)

peptide probe 1 peptide probe 2 peptide probe 3 peptide probe 4 peptide probe 1

profilin profilin profilin profilin profilin blocked with peptide probe 1

633 422 219 93 158

of 158 pN, the highest peak of the histogram is at around 60 pN. Since the highest peaks for peptide probes 2 and 3 were also near 60 pN, the nonspecific binding force of peptide probe 1 is at around 60 pN. In Table 1, the averaged forces show that, of the four peptide probes examined, peptide probe 1 has the highest affinity for profilin. The results support a specific interaction of peptide probe 1 with profilin. Peptide probe 4 has the smallest retraction force (Table 1). Since peptide probe 4 does not contain the GP5 sequence, the retraction force for peptide probe 4 (93 pN) is supposed to represent the nonspecific binding force. This sequence-dependent affinity was also demonstrated when comparing the retraction forces of peptide probes 2 and 3. Nevertheless, although peptide probes 2 and 3 are composed of the same number of amino acids, the averaged retraction force of peptide probe 2 was approximately twice as large as that of peptide probe 3. This large retraction force is reasonable, since peptide probe 2 includes the GP5 sequence that represents one segment of the binding sequence of VASP to profilin (with three consecutive GP5 sequences). While the previous study reported low affinity of a single repeat peptide (GP5) to profilin, the averaged retraction force of GP5 (peptide probe 2) in this study is large. This relatively large force would result if peptide probe (23) Jonckheere, V.; Lambrechts, A.; Vandekerckhove, J.; Ampe, C. FEBS Lett. 1999, 447, 257-263. (24) Okada, T.; Yamamoto, Y.; Miyachi, H.; Karube, I.; Muramatsu, H. Biosens. Bioelectron. 2006, 22, 1480-1486. (25) Wagner, P.; Hegner, M.; Kernen, P.; Zaugg, F.; Semenza, G. Biophys. J. 1996, 70, 2052-2066. (26) Flach, C. R.; Prendergast, F. G.; Mendelsohn, R. Biophys. J. 1996, 70, 539-546.

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Figure 5. Histograms of the detailed retraction forces (without maximum force for each curve) between profilin and (a) peptide probe 1, (b) peptide probe 2, (c) peptide probe 3, (d) peptide probe 4, and (e) between blocked profilin and peptide probe 1. Histograms are represented in probability (%) vs force (pN). Solid line in histogram (a) represents a Gaussian fitting curve.

2 was immobilized at high density and where more than a probe molecule bound to profilin concurrently. The averaged retraction force of peptide probe 1 (633 pN) would include multiple interactions rather than just a single molecule interaction. Under these experimental conditions, multiple interactions consistently occur, since the estimated contact area between the mica surface and the cantilever tip (see the Supporting Information) corresponds to tens of profilin molecules, as the occupation area for a single profilin molecule is ∼9 nm2.27 To verify the retraction force of a single molecular interaction, the obtained force curve was analyzed further. For example, the force curve shown in Figure 4a shows a maximum retraction force at 625 pN, followed by several small retraction forces around 80 pN. Since these small retraction forces are supposed to represent a single molecule interaction between peptide probe 1 and profilin, the small retraction forces for each peptide probe 1-4 were collected and summarized in the histograms (Figure 5). These histograms show the probability of small retraction forces without the maximum retraction forces in each force curve; that is, the histograms in Figure 5 exclusively represent the small retraction forces in the latter course of the retraction step, and the shapes of the histograms completely differ from those shown in the Supporting Information. For peptide probe 1, the first peak appears at around 50 pN, followed by several peaks up to around 600 pN which represent multiple interactions. In an effort to analyze the highest peak positions, the histogram for peptide probe 1 (Figure 5a) was fitted to a Gaussian curve. For the Gaussian fitting process, forces in the range of 60-150 pN were used. Forces smaller than 60 pN were omitted because peptide probe 3 (Figure 5c) exhibits the highest peak at around 60 pN, and the control experiment shown in the Supporting Information also suggests that the nonspecific binding of peptide probe 1 to profilin is at around 80 pN. Forces larger than 150 pN were also omitted, since the histogram exhibits the next peak at around 150 pN. The optimized Gaussian curve

gives the peak position at 96 pN, and the curve is shown in Figure 5a. The peak position allows an estimate of 96 pN for the single molecule retraction force between peptide probe 1 and profilin. The single molecule interaction force obtained is similar to the value previously reported for forces between proteins.6,28

(27) Nodelman, I. M.; Bowman, G. D.; Lindberg, U.; Schutt, C. E. J. Mol. Biol. 1999, 294, 1271-1285.

(28) Allen, S.; Davies, J.; Davies, M. C.; Dawkes, A. C.; Roberts, C. J.; Tendler, S. J.; Williams, P. M. Biochem. J. 1999, 341, 173-178.

Conclusions Four different peptide probes 1-4 were synthesized and evaluated in relation to their affinities for profilin by measuring the requisite force curves. Each peptide probe was immobilized to the cantilever tip, and profilin was covalently immobilized to the mica substrate. Immobilization of the peptide probe was characterized by FT-IR spectroscopy and XPS, and that of profilin was confirmed by examination of AFM topographic images. Analysis of the results derived from the force curves indicated that peptide probe 1 showed the largest retraction force. The large retraction force indicates high affinity of the GP5GP5GP5 moiety for profilin, and the small effect derived from the linker moiety was confirmed by the smallest retraction force being recorded for peptide probe 4. These results confirmed the sequence-dependent affinity of the peptide probe for profilin. The single molecule retraction force was also estimated by detailed analyses of the force curves. This study revealed that the single molecule retraction force between peptide probe 1 and profilin is 96 pN. This method for estimating single molecule interactions can be applied to the evaluation of the affinity of various biomolecules. Since force curve measurements represent a powerful technique to analyze small localized areas, our future work will include an evaluation of protein interactions at the cell surface using peptide probes. Acknowledgment. This research was partly supported by the New Energy and Industrial Technology Development Organization (NEDO).

Binding Affinity of Peptide Probes for Profilin

Supporting Information Available: Calibration method of the spring constant, FT-IR spectra in the full range, a topographic image of the profilin-immobilized mica substrate, estimation of the contact area between the cantilever tip and the substrate, and histograms of the

Langmuir, Vol. 24, No. 8, 2008 4055 maximum retraction forces observed between profilin and the peptide probes. This material is available free of charge via the Internet at http://pubs.acs.org. LA703344U