Self-Assembly of Nanofiber with Uniform Width from Wheel-Type

Feb 21, 2008 - A novel C3 symmetric peptide conjugate “Wheel-FKFE” consisting of three β-sheet-forming peptides with wheel-like arrangement is de...
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Biomacromolecules 2008, 9, 913–918

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Self-Assembly of Nanofiber with Uniform Width from Wheel-Type Trigonal-β-Sheet-Forming Peptide Kazuya Murasato,† Kazunori Matsuura,*,†,‡ and Nobuo Kimizuka† Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu University, Moto-oka 744, Nishi-ku, Fukuoka 819-0395, Japan, and PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama, 332-0012, Japan Received November 26, 2007; Revised Manuscript Received December 25, 2007

A novel C3 symmetric peptide conjugate “Wheel-FKFE” consisting of three β-sheet-forming peptides with wheellike arrangement is developed, and the morphology of self-assembled peptide conjugates in aqueous solutions is observed at various pH. The CD spectra of Wheel-FKFE show the formation of β-sheet structures in pH 6.9 phosphate buffer, whereas random structures are formed in aqueous HCl (pH 3.3) and NaOH (pH 11) solutions. In transmission electron microscopy, nanofibers with a uniform width of 3–4 nm and lengths of several micrometers are observed in pH 6.9 phosphate buffer, whereas nanorods with the width of several nanometers and the length of several tens of nanometers are observed for that of aqueous HCl (pH 3.3) and NaOH (pH 11) solutions. The uniform width (3–4 nm) of the fibers observed in neutral solution indicates formation of columnar self-assembly of Wheel-FKFEs. The fluorescence spectrum of polarity sensitive dye, sodium 8-anilino-1-naphthalenesulfonate (ANS), in the presence of Wheel-FKFE fibers revealed that the polarity inside the fibers corresponds to that of acetone, indicating that the internal space of the fibers possesses medium hydrophobic environment.

Introduction In biological systems, many kinds of supramolecular assemblies of proteins play pivotal roles. Many protein assemblies are built by interactions between secondary structures, such as R-helixes and β-sheets, at the interface of unit proteins.1 For example, capsule-like protein assemblies such as ferritin2 and clathrins3 are built by interactions between R-helixes. The internal skeleton of tomato bushy stunt virus is built by the formation of intermolecular β-sheets among C3 symmetric protein units.4 Amyloid fibrils deposited in the brains of Alzheimer’s patients consist of extraordinarily extended β-sheets.5 Artificial supramolecular assemblies consisting of coiled-coil R-helix peptides6 and ionic complementary β-sheet-forming peptides7 have been designed. Especially, self-assembly of β-sheet-forming peptides have attracted much attention not only as model compounds to reveal the mechanism of Alzheimer’s disease but also as functional biomaterials8 such as scaffolds for tissue engineering9 and one-dimensional molecular arrays.10 Gazit et al. reported that even aromatic dipeptides self-assembled into nanotubes and nanospheres.11 Recently, it was reported that preorganization of the spatial arrangement of peptides afford unique assemblies depending on the molecular structures. For example, disulfide-bridged β-turn peptides self-assembled into capsid-like nanotubes,12 and ferrocene-conjugated peptides were forced into β-conformation.13 Branched conjugate of coiled-coil-forming peptides formed branched nanofibers,14 while dendrimers with a leucinezipper unit self-assembled into helical fibrils.15 Such preorganized peptide conjugates can be designed to form higher-order structures and show functions peculiar to the structures. * To whom correspondence should be addressed. Phone: +81 92 802 2833. Fax: +81 92 802 2838 . E-mail: [email protected]. † Kyushu University. ‡ PRESTO, Japan Science and Technology Agency.

Scheme 1. Synthesis of Wheel-FKFE

We have developed a novel strategy for construction of spherical assemblies consisting of biomolecules such as DNA16 and peptides.17 DNA three-way junctions bearing self-complementary sticky ends were self-assembled into nanometer to micrometer sized spherical structures depending on the concentration.16 Artificial C3 symmetric radial peptide conjugates (Trigonal(FKFE)2, Chart 1) were self-assembled into capsulelike structures with a size of about 20 nm via formation of antiparallel β-sheets in the acidic aqueous solution.17 The C3 symmetric preorganization of β-sheet-forming peptide chains might reduce the entropy loss in the self-assembling process

10.1021/bm701302p CCC: $40.75  2008 American Chemical Society Published on Web 02/21/2008

914 Biomacromolecules, Vol. 9, No. 3, 2008 Chart 1.

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Structure of Trigonal(FKFE)2

and afford unique assemblies which short β-sheet-forming peptides themselves cannot form. Similar molecular designs of C3 symmetric peptide conjugates were reported recently by other groups. It was reported that C3 symmetrically linked dipeptides can form vesicles18 and columnar assemblies.19 C3 symmetric helical peptide conjugates were designed to regulate the molecular conformation utilizing the dipole–dipole interactions.20 In this paper, we demonstrate the self-assembly of a novel C3 symmetric peptide conjugate consisting of three β-sheetforming peptide conjugates, named Wheel-FKFE, with wheellike arrangement. Wheel-FKFE possesses three peptides FKFECKFE connected to arm of C3 symmetric core at the C residue placed at the middle of sequence, whose core and peptides are considered as hub and spokes of a wheel, respectively (Scheme 1). Wheel-FKFE was designed as a platelike molecule, which is expected to be assembled into novel nanostructures by formation of intermolecular β-sheets.

Results Synthesis of Wheel-FKFE. Wheel-FKFE was synthesized according to Scheme 1. Melamine unit was adopted as a C3 symmetric core to improve the hydrophilicity compared to the benzene unit employed in the previously reported C3 symmetric peptide conjugate Trigonal(FKFE)2.17 The C3 symmetric core 2 was synthesized by tris-substitution of cyanuric chloride with N-Boc-ethylenediamine followed by deprotection and iodoacetylation. The 8-mer peptide H-FKFECKFE-OH was designed based on β-sheet-forming peptide (FKFEFKFE) reported by Zhang et al.7e and synthesized by standard Fmoc-protecting solid-phase method. The C-residue was placed on the center position of the sequence to construct wheel-like arrangement of peptides. Wheel-FKFE was prepared by coupling of thiol groups of the 8-mer precursor peptides with C3 symmetric iodoacetoamidated core molecules 2, purified by reversed-phase HPLC and confirmed by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF-MS). (m/z ) 3607, [M + H]+). The purified Wheel-FKFE dissolved well in aqueous media at whole range of pH conditions, whereas the previously reported Trigonal(FKFE)2 was soluble only in acidic water. Secondary structure of Wheel-FKFE. A circular dichroism (CD) spectrum of Wheel-FKFE showed a positive peak at 195 nm and a negative peak at 219 nm in phosphate buffer (pH 6.9), indicating the formation of typical β-sheet structures

Figure 1. CD spectra of aqueous solution of (A) Wheel-FKFE (31 µM) and (B) FKFECKFE (93 µM) at 25 °C (inset: difference CD spectrum obtained by subtracting the spectrum at pH 6.9 from that at pH 3.3).

(Figure 1A).21 Conversely, in aqueous HCl (pH 3.3) and aqueous NaOH (pH 11) solution, CD spectra of Wheel-FKFE showed a weak positive peak at 220 nm and a weak negative peak at 200 nm, indicating random-coil structures. The CD spectra of the precursor peptide H-FKFECKFE-OH showed that the peptide adopted random-coil structure in aqueous HCl (pH 3.3) and aqueous NaOH (pH 11) solution, whereas at pH 6.9 a certain amount of β-sheet component was seemingly present in phosphate buffer (Figure 1B). Self-Assembly of Wheel-FKFE in Water. A transmission electron microscopy (TEM) image revealed that Wheel-FKFE formed nanofibers of several micrometers length in phosphate buffer (pH 6.9, Figure 2a,b), whereas it formed rodlike structures in aqueous HCl (pH 3.3, Figure 2c) and NaOH (pH 11, Figure 2d) solutions. Interestingly, the observed fibers at pH 6.9 possess a fairly uniform width of 3–4 nm, which corresponded to the single-molecular size of Wheel-FKFE. The more extended fibrous assemblies were observed for pH 6.9 phosphate buffer solutions which exhibited a β-sheet-rich CD spectrum (Figure 1A). These results suggest that the extended fibrous assemblies are comprised of β-sheet structures. Fibrous structures were also obtained from a solution of the precursor peptide H-FKFECKFE-OH at pH 6.9 (Figure 2e,f). However, the fibers from the precursor peptides were tapelike and spirally twisted structures with width distributed from 4 to 25 nm, which is different from fibers of Wheel-FKFEs with uniform width.

Self-Assembly of Wheel-Type Peptides

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Figure 4. Fluorescence spectra of ANS (100 µM) in pH 6.9 phosphate buffer, λex ) 380 nm: green, ANS alone; red, with Wheel-FKFE (28 µM); blue, with FKFECKFE (84 µM).

Figure 2. TEM images of negative stained Wheel-FKFE and precursor peptide FKFECKFE. (a, b) [Wheel-FKFE] ) 31 µM in phosphate buffer (pH 6.9) stained by phosphotungstic acid. (c) [Wheel-FKFE] ) 31 µM in aqueous HCl (pH 3.3) stained by uranyl acetate. (d) [Wheel-FKFE] ) 31 µM in aqueous NaOH (pH 11) stained by uranyl acetate. (e, f) [FKFECKFE] ) 93 µM in phosphate buffer (pH 6.9) stained by phosphotungstic acid.

Figure 3. Fluorescence spectra of ThT (100 µM) in pH 6.9 phosphate buffer, λex ) 450 nm: green, ThT alone; red, with Wheel-FKFE (28 µM); blue, with FKFECKFE (84 µM).

Binging of Thioflavin T and ANS to the Assembly of Wheel-FKFE. Thioflavin-T (ThT) is a fluorescent dye whose intensity at 490 nm (excitation 450 nm) is enhanced by binding to β-sheets assemblies.22 Figure 3 shows fluorescence spectra of ThT in the presence of Wheel-FKFE and the precursor peptide in phosphate buffer (pH 6.9). The fluorescence intensity of ThT was remarkably enhanced in the presence of WheelFKFE (about 271-fold as compared with the spectrum of ThT alone), while it is only slightly enhanced in the presence of the precursor peptide. Sodium 8-anilino-1-naphthalenesulfonate (ANS), which is a polarity-sensitive fluorescence dye, was used to investigate the polarity inside the Wheel-FKFE fibers. The fluorescence spectrum of ANS in the presence of Wheel-FKFE fibers in

phosphate buffer (pH 6.9) exhibited an intense peak at 462 nm (Figure 4), which was blue-shifted, and the intensity was increased about 14-fold as compared with the spectrum of ANS alone in phosphate buffer. The blue shift of the emission peak of ANS occurred also in the presence of precursor peptide H-FKFECKFE-OH; however, the shift and the increment of intensity were smaller than those in the presence of WheelFKFE.

Discussion As described above, the novel C3 symmetric peptide conjugate Wheel-FKFE formed β-sheet structure in neutral phosphate buffer, which was self-assembled into nanofibers with singlemolecular thickness. In contrast, random structures were formed in acidic and basic aqueous solutions (Figures 1A and 2a-d). Here we propose a model structure of self-assembly of WheelFKFE in neutral solution (Figure 5). The Wheel-FKFE molecule consists of hydrophilic periphery (K and E residues, blue part) and hydrophobic interior (F residues and triazine core, yellow part). The fibrous assemblies with uniform width might be constructed by the stacking of wheels via hydrophobic moieties and intermolecular β-sheets. As the fibers display hydrophilic zwitterions to the aqueous phase and hydrophobic moieties inside, irregular aggregation or bundling of fibers by hydrophobic association are suppressed. The pH dependence of the secondary and self-assembled structure of Wheel-FKFE is probably ascribed to the difference of peptide charges. The peptide H-FKFECKFE-OH possesses three amino groups and three carboxyl groups, respectively. At pH 6.9, the total charge of the peptide is neutralized. Reduction of electrostatic repulsion between peptides promotes the formation of β-sheets and the extended fibrous assemblies. In contrast, the total charge of the peptide is positive at pH 3.3 or negative at pH 11, which causes electrostatic repulsions and formation of β-sheets and fibrous assemblies are suppressed. The precursor peptide H-FKFECKFE-OH showed smaller molar ellipticity (at 219 nm) compared to that of Wheel-FKFE in neutral phosphate buffer (Figure 1B), indicating that WheelFKFE possesses richer β-sheet content than those of HFKFECKFE-OH at the pH. It is therefore speculated that the formation of β-sheets of the peptide FKFECKFE is promoted by the C3 symmetric preorganization. In the TEM image of the precursor peptide at pH 6.9, tapelike and spirally twisted structures with width distributed from 4 to 25 nm are abundantly

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contrast, the peptide units of Wheel-FKFEs in nanofibers form antiparallel β-sheets with attractive intermolecular ionic complementarity. Moreover, Wheel-FKFE has the potential to limit its conformation to planar triangle structure. Since each peptide has a cationic N-terminal and an anionic C-terminal, they will form intramolecular head-to-tail electrostatic interactions (salt bridges) which stabilize the peptide triangle.20 The planarity of Wheel-FKFE can also promote the stacking assembly.

Conclusion We have demonstrated that novel C3 symmetric peptide conjugate, Wheel-FKFE, self-assembled into β-sheet nanofibers with uniform, single-molecular-sized width in neutral phosphate buffer. It was found that the nanofibers are formed by stacking and hydrogen bonding between the molecules, which possesses extended β-sheet structure and hydrophobic space in the fibers. The molecular design to regulate the geometry of β-sheetforming peptides by tethering to a symmetric junction molecule can be widely extended to the construction of various nanostructures. We expect that the assemblies would be developed into functional nanomaterials utilizing their own shapes.

Experimental Section

Figure 5. Schematic illustration of the self-assembly of Wheel-FKFEs.

seen (Figure 2e,f). Since the hydrophobic moieties of the precursor peptide are probably exposed to water, the peptide undergoes bundling to tapelike structures without control on the hydrophobic aggregation. The results of ThT binding (Figure 3) indicate that WheelFKFEs form ordered assemblies with higher β-sheet content, whereas the precursor peptides form less ordered assemblies with lower β-sheet content. These results corresponded to the observed difference of CD spectra between Wheel-FKFE and H-FKFECKFE-OH. The results of ANS binding (Figure 4) suggest that the Wheel-FKFE nanofibers possess lower polarity than near the H-FKFECKFE-OH fibers. The peak wavelength of ANS fluorescence in the presence of Wheel-FKFEs is correlated with the polarity scale (ETN value) of solvent.23 According to the relationship, the polarity of the Wheel-FKFE nanofibers corresponds to an ETN value of 0.4, which is comparable to that in acetone solution. The hydrophobic space incorporating ANS might exist between the layers of WheelFKFEs or at the apertures between the C3 symmetric core and peptide chains. These results are well explained by the selfassembly model of Wheel-FKFEs illustrated in Figure 5. Finally, we discuss the difference between the previously reported Trigonal(FKFE)2 (Chart 1, spherical assemblies)17 and the present Wheel-FKEE (fibrous assemblies). These difference may be attributed to the ionic complementarity of positively charged amino groups (K residues) and negatively charged carboxylate groups (E residues).7c If Trigonal(FKFE)2s form nanofibers, they would have aligned to form parallel β-sheets against the repulsive ionic complementarity. This makes the formation of nanofibers of Trigonal(FKFE)2 unfavorable. In

General Information. Reagents and solvents were obtained from commercial sources and used without further purification unless indicated. 1H NMR spectra were recorded on Bruker DRX 600 spectrometer at ambient temperature with tetramethylsilane or residual solvent peak as internal references. IR spectra were recorded with a Shimadzu IRPrestige-21 spectrophotometer. CD spectra were taken at 25 °C in a 1.0 mm quartz cell with a JASCO J-820 spectrophotometer equipped with a Peltier-type thermostatic cell holder. Reversed-phase HPLC was performed at ambient temperature with a Simadzu LC-6AD liquid chromatograph equipped with a UV-vis detector (220 nm, Shimadzu SPD-10AVvp) using GL Science Inertsil ODS-3 columns (4.6 × 250 mm and 20 × 250 mm). MALDI-TOF mass spectra were obtained on a PE Applied Biosystems Voyager System 1180 MALDITOF type mass spectrometer with dithranol and R-cyano-4-hydroxycinnamic acid (R-CHCA) as matrix. Transmission electron microscopy (TEM) was conducted on a JEOL-2010 instrument operated at 120 kV with uranyl acetate (2 wt %) or phosphotungstic acid (2 wt %) as a negative stainer. Solid Phase Synthesis of Peptide H-FKFECKFE-OH. H-PheLys(Boc)-Phe-Glu(OtBu)-Cys(Trt)-Lys(Boc)-Phe-Glu(OtBu)-Alko resin was synthesized by stepwise elongation of N-Fmoc-R-amino acids (3 equiv) on R-p-alkoxybenzyl alcohol resin (Alko resin, Watanabe Chemical Ind. Ltd., 0.60 mmol/g) with 2-(1H-benzotriazole-1-yl)1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU, 3 equiv), 1-hydroxybenzotriazole hydrate (HOBt · H2O, 3 equiv), and N,Ndiisopropylethylamine (DIPEA, 3 equiv) in N-methyl-2-pyrrolidone (NMP) as a coupling reagent, and 20% piperidine in NMP for Fmoc deprotection. The peptidyl-resin was washed with NMP, dichloromethane, and methanol and then dried under vacuum. The peptides were deprotected and cleaved from the resin by treatment with a cocktail of TFA/water/1,2-ethanedithiol/triisopropylsilane ) 94/2.5/2.5/1 in volume at room temperature for 3 h. The reaction mixture was filtered to remove the resin, and the filtrate was concentrated under vacuum. The peptide was precipitated by adding ice-cooled methyl tert-butyl ether (MTBE) to the residue, and the supernatant was decanted. After the MTBA washing was repeated three times, the precipitated peptide was dried under vacuum. The crude product was purified by C18reversed-phase HPLC eluting with a liner gradient of CH3CN/water (20/80 to 30/70 over 40 min) containing 0.1% TFA. The elution fraction containing the desired peptide was lyophilized to give a flocculent solid. The isolated yield was 60%. MALDI-TOF-MS (matrix:

Self-Assembly of Wheel-Type Peptides R-CHCA): m/z calcd for C52H72N10O13S, 1076.50 [M + H]+; found, 1077.06 [M + H]+. Synthesis of 2,4,6-Tris[2-(N-tert-butoxycarbonyl)ethylamino)]1,3,5-triazine (1). A solution of cyanuric chloride (0.41 g, 2.5 mmol) in toluene (40 mL) was added to a solution of N-(2-aminoethyl)carbamic acid tert-butyl ester (2.4 g, 15 mmol) and DIPEA (1.9 g, 15 mmol) in toluene (20 mL) at room temperature. The mixture was refluxed for 9 h, and the solvent was evaporated. The residue was dissolved in chloroform (100 mL) and washed with 0.1 M aqueous HCl (3 × 30 mL) and deionized water (10 × 20 mL). The organic layer was dried over anhydrous Na2SO4 and evaporated to a brown solid. The residue was washed with toluene followed by n-hexane to provide pure compound 1 (1.2 g, 84%). 1H NMR (600 MHz, CDCl3): δ 5.3–5.8 (br m, 6H), 3.46 (br s, 6H), 3.30 (br s, 6H), 1.42 (br s, 27H). FT-IR (KBr, cm-1) 3462, 3435, 3277, 3156, 2980, 2937, 1690, 1570, 1531, 1253, 1177, 1124, 1066. Synthesis of 2,4,6-Tris[2-(iodoacetoamido)ethylamino]-1,3,5-triazine (2). Trifluoroacetic acid (1.0 mL, 13 mmol) and deionized water (50 µL) was added to a suspension of compound 1 (0.11 g, 0.21 mmol) in dichloromethane (2.0 mL). After the mixture was stirred for 1 h at room temperature, excess trifluoroacetic acid and dichloromethane was evaporated to a colorless sticky solid. The residue was dissolved in 0.6 M aqueous NaHCO3 and added a solution of iodoacetic acid N-hydroxy succinimide ester (0.29 g, 1.2 mmol) in acetone (3.0 mL). The mixture was stirred in the dark for 1 h at room temperature and the resulting precipitate was filtered, washed with water and acetone, and dried under vacuum to provide crude compound 2 (48 mg, 30%). The product was not purified further and was used in the next reaction. MALDI-TOF-MS (matrix: R-CHCA): m/z calcd for C15H24I3N9O3, 758.91 [M + H]+; found, 759.65 [M + H]+. Synthesis of the Peptide Conjugate Wheel-FKFE (3). Peptide H-FKFECKFE-OH (4.7 mg, 4.4 µmol) was dissolved in degassed DMF (1.0 mL). To the solution was added a mixture of diidopropylethylamin (4.5 mg, 35 µmol), 2,2,2-trifluoroethanol (0.10 mL), and DMF (1.0 mL) under nitrogen atmosphere. DMF solution (1.0 mL) of 2 (1.0 mg, 1.3 µmol) was added to the mixture at -20 °C. The reaction mixture was stirred in the dark at the same temperature for 51 h. In the course of the reaction, additional diisopropylethylamine (3.4 mg, 26 µmol) in DMF (0.50 mL), degassed water (2.0 mL), and additional peptide (2.0 mg, 1.9 µmol) in water (1.0 mL) were added. The reaction mixture was lyophilized to a colorless solid. The crude product was purified by reversed-phase HPLC eluting with a liner gradient of CH3CN/water (23/77 to 28/72 over 40 min) containing 0.1% TFA. The elution fraction containing 3 was lyophilized to a flocculent solid (1.25 mg, 25%). MALDI-TOFMS (matrix: R-CHCA): m/z calcd for C171H237N39O42S3, 3607.68 [M + H]+; found, 3607.37 [M + H]+. 1H NMR (600 MHz, D2O): δ7.1–7.3 (br m, 45H), 4.6 (br m, 3H), 4.5 (br m, 3H), 4.2–4.4 (br m, 21H), 3.3–3.5 (br m, 18H), 3.2 (br m, 6H), 3.1 (br m, 9H), 2.8–3.0(br m, 18H), 2.3 (br t, 12H), 1.5–1.7 (br m, 24H), 1.1–1.5 (br m, 24H). Transmission Electron Microscopy (TEM). One drop of sample solutions was placed on a carbon-coated Cu grid (Oken Co., Ltd.) at room temperature. The droplet was left for 60 s and then removed. Subsequently, the staining solution (2% uranyl acetate for aqueous HCl and NaOH solutions, 2% phosphotangstic acid for phosphate buffer solutions) was placed on the grid and removed immediately. The stained grid was dried in vacuo (poststaining method). The sample grids were subjected to observation on a JEM-2010 instrument (JEOL) with an acceleration voltage of 120 kV. Binding of Thioflavin-T (ThT) to Wheel-FKFE. Aqueous solutions containing Wheel-FKFE (28 µM) or H-FKFECKFE-OH (84 µM) and ThT (0.10 mM) were prepared and measured the fluorescence spectra of ThT with excitation at 450 nm at 25 °C by a HITACHI F-4500 instrument.

Biomacromolecules, Vol. 9, No. 3, 2008 917 Binding of 8-Anilino-1-naphthalenesulfonic Acid (ANS) to Wheel-FKFE. Aqueous solutions containing Wheel-FKFE (28 µM) or H-FKFECKFE-OH (84 µM) and ANS (0.10 mM) were prepared and measured the fluorescence spectra of ANS with excitation at 380 nm at 25 °C by a HITACHI F-4500 instrument.

Acknowledgment. This present work is supported by a fund from Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), and a Grant-in-Aid for the Global COE Program, “Science for Future Molecular Systems” from the Ministry of Education, Culture, Science, Sports and Technology of Japan.

References and Notes (1) Branden, C.; Tooze, J. Introduction to Protein Structure, 2nd ed.; Garland Publishing: New York, 1999. (2) Lawson, D. M.; Artymiuk, P. J.; Yewdall, S. J.; Smith, J. M. A.; Livingstone, J. C.; Treffry, A.; Luzzago, A.; Levi, S.; Arosio, P.; Cesareni, G.; Thomas, C. D.; Shaw, W. V.; Harrison, P. M. Nature 1991, 349, 541. (3) Fotin, A.; Cheng, Y.; Sliz, P.; Grigorieff, N.; Harrison, S. C.; Kirchhausen, T.; Walz, T. Nature 2004, 432, 573. (4) (a) Olson, A. J.; Bricogne, G.; Harrison, S. C. J. Mol. Biol. 1983, 171, 61. (b) Hopper, P.; Harrison, S. C.; Sauer, R. T. J. Mol. Biol. 1984, 177, 701. (5) (a) McLaurin, J.; Yang, D.-S.; Yip, C. M.; Fraser, P. E. J. Struct. Biol. 2000, 130, 259. (b) Jiménez, J. L.; Nettleton, E. J.; Bouchard, M.; Robinson, C. V.; Dobson, C. M.; Saibil, H. R. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9196. (6) (a) MacPhee, C. E.; Woolfson, D. N. Curr. Opin. Solid State Mater. Sci. 2004, 8, 141. (b) Pandya, M. J.; Spooner, G. M.; Sunde, M.; Thorpe, J. R.; Rodger, A.; Woolfson, D. N. Biochemistry 2000, 39, 8728. (c) Liu, J.; Yong, W.; Deng, Y.; Kallenbach, N. R.; Lu, M. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 16156. (7) (a) Pastor, M. T.; Esteras-Chopo, A.; Paz, M. L. Curr. Opin. Struct. Biol. 2005, 15, 57. (b) Mihara, H.; Matsumura, S.; Takahashi, T. Bull. Chem. Soc. Jpn. 2005, 78, 572. (c) Chen, P. Colloids Surf., A 2005, 261, 3. (d) Zhang, S.; Holmes, T.; Lockshin, C.; Rich, A. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 3334. (e) Marini, D. M.; Hwang, W.; Lauffenburger, D. A.; Zhang, S.; Kamm, R. D. Nano Lett. 2002, 2, 295. (f) Aggeli, A.; Bell, M.; Carrick, L. M.; Fishwick, C. W. G.; Harding, R.; Mawer, P. J.; Radford, S. E.; Strong, A. E.; Boden, N. J. Am. Chem. Soc. 2003, 125, 9619. (8) (a) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128. (b) Zhang, S. Nat. Biotechnol. 2003, 21, 1171. (c) Fairman, R.; Åkerfeldt, K. S. Curr. Opin. Struct. Biol. 2005, 15, 453. (d) Gao, X.; Matsui, H. AdV. Mater. 2005, 17, 2037. (9) (a) Kisiday, J.; Jin, M.; Kurz, B.; Hung, H.; Semino, C.; Zhang, S.; Grodzinsky, A. J. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9996. (b) Ellis-Behnke, R. G.; Liang, Y.-X.; You, S.-W.; Tay, D. K. C.; Zhang, S.; So, K.-F.; Schneider, G. E. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 5054. (c) Salick, D. A.; Kretsinger, J. K.; Pochan, D. J.; Schneider, J. P. J. Am. Chem. Soc. 2007, 129, 14793. (10) (a) Kodama, H.; Matsumura, S.; Yamashita, T.; Mihara, H. Chem. Commun. 2004, 2876. (b) Matsumura, S.; Uemura, S.; Mihara, H. Mol. BioSyst. 2005, 1, 146. (11) (a) Reches, M.; Gazit, E. Nano Lett. 2004, 4, 581. (b) Hendler, N.; Sidelman, N.; Reches, M.; Gazit, E.; Rosenberg, Y.; Richter, S. AdV. Mater. 2007, 19, 1485. (c) Reches, M.; Gazit, E. Science 2003, 300, 625. (12) Valéry, C.; Paternostre, M.; Robert, B.; Gulik-Krzywicki, T.; Narayanan, T.; Dedieu, J. C.; Keller, G.; Torres, M. L.; Cherif-Cheikh, R.; Calvo, P.; Artzner, F. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 10258. (13) Barišic´, L.; Cˇakic´, M.; Mahmoud, K. A.; Liu, Y.; Kraatz, H.-B.; Pritzkow, H.; Kirin, S. I.; Metzler-Nolte, N.; Rapic´, V. Chem. Eur. J. 2006, 12, 4965. (14) Ryadnov, M. G.; Woolfson, D. N. J. Am. Chem. Soc. 2005, 127, 12407. (15) Zhou, M.; Bentley, D.; Ghosh, I. J. Am. Chem. Soc. 2004, 126, 734. (16) (a) Matsuura, K.; Yamashita, T.; Igami, Y.; Kimizuka, N. Chem. Commun. 2003, 376. (b) Kim, K.; Masumoto, K.; Matsuura, K.; Kimizuka, N. Chem. Lett. 2006, 35, 486. (c) Kim, K.; Matsuura, K.; Kimizuka, N. Bioorg. Med. Chem. 2007, 15, 4311. (d) Matsuura, K.; Masumoto, K.; Igami, Y.; Fujioka, T.; Kimizuka, N. Biomacromolecules 2007, 8, 2726. (17) Matsuura, K.; Murasato, K.; Kimizuka, N. J. Am. Chem. Soc. 2005, 127, 10148.

918 Biomacromolecules, Vol. 9, No. 3, 2008 (18) Ghosh, S.; Reches, M.; Gazit, E. Verma S. Angew. Chem., Int. Ed. 2007, 46, 2002. (19) van den Hout, K. P.; Martín-Rapún, R.; Vekemans, J. A. J. M.; Meijer, E. W. Chem. Eur. J. 2007, 13, 8111. (20) Ishikawa, T.; Morita, T.; Kimura, S. Bull. Chem. Soc. Jpn. 2007, 80, 1483. (21) Greenfield, N. J. Anal. Biochem. 1996, 235, 1.

Murasato et al. (22) (a) Krebs, M. R. H.; Bromley, E. H. C.; Donald, A. M. J. Struct. Biol. 2005, 149, 30. (b) Groenning, M.; Olsen, L.; van de Weert, M.; Flink, J. M.; Frokjaer, S.; Jørgensen, F. S. J. Struct. Biol. 2007, 158, 358. (23) Hayashida, O.; Ono, K.; Hisaeda, Y.; Murakami, Y. Tetrahedron 1995, 51, 8423.

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