Phased Fiber Growth in a Peptide Conjugate: Aggregation and

Mar 21, 2007 - concentration-dependent fashion. Ultrastructural details of ... acids were purchased from Spectrochem, Mumbai, India and used without f...
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3750

J. Phys. Chem. B 2007, 111, 3750-3757

Phased Fiber Growth in a Peptide Conjugate: Aggregation and Disaggregation Studies Surajit Ghosh and Sandeep Verma* Department of Chemistry, Indian Institute of Technology-Kanpur, Kanpur-208016 (UP), India ReceiVed: October 5, 2006; In Final Form: January 27, 2007

A glycine-rich, short pentapeptide conjugate 6, derived from the highly conserved copper-binding octarepeat region of the prion protein, exhibits a tendency to self-aggregate in a time-dependent fashion. Aging of 6 afforded an insight into the phased growth of spherical prefibrillar structures to fibers of long persistence length, as observed by a combination of microscopic techniques. Interestingly, growth of these fibers was inhibited by colchicine, a known inhibitor of microtubule polymerization in a concentration dependent fashion. This study offers an intriguing insight into the occurrence of prefibrillar intermediates on the path to the formation of full length peptide fibers. It is also envisaged that constructs such as 6 may also serve as simple models to study chemical intervention of protein aggregation.

Introduction Protein aggregation is closely involved with the etiology of several neurodegenerative diseases.1 This has spurred widespread interest in understanding the fundamental aspects involved with protein misfolding and events leading to this catastrophic aggregative phenomenon that causes loss of cognitive functions.2 It is now believed that this process is highly dependent on intermolecular interactions of polypeptide chains, further aided by other peripheral interactions, which stabilize prefibrillar intermediates eventually leading to highly amorphous and protease resistant proteinaceous aggregates.3 In this context, it has also been suggested that aggregative propensities of amyloidogenic proteins may reside in small peptide fragments that can exhibit agglomeration under experimental conditions.4 Thus, it is obvious that the misfolding phenomenon requires considerable understanding of molecular interactions that govern aggregative processes in amyloidogenic and non-amyloidogenic proteins. However, a major lacuna in such studies originates from the highly amorphous nature of aggregated proteins, which prevents determination of detailed atomic structure by conventional diffraction methods, and moreover, the insoluble nature of these aggregates prohibits solution-phase studies. In this context, recent reports dealing with HETs and the Sup35p prion protein have provided crucial insight into the structural organization and requirements for the fibril formation.5 PrPC, the normal cellular form of the prion protein, is predominantly R-helical, and it misfolds into a β-sheet rich, pathogenic isoform (PrPSc) subsequent to key mutations in the structured C-terminal domain, causing drastic reorganization of secondary and tertiary structural features (Figure 1).6 In contrast, the N-terminal domain of the prion protein (aa 29-124) is largely unstructured and conformationally labile but contains four evolutionary conserved peptide octarepeats that are implicated for copper binding.7,8 The presence of a repetitive metal binding motif suggests that the prion protein may serve as a periplasmic copper transporter and may be involved with other copper-dependent activities. These proteins are fascinating for several reasons including their ability to adopt deviant “infectious” conformation states leading to self-perpetuation, protein* Email: [email protected].

Figure 1. Schematic representation of the prion protein indicating locations of structured/unstructured regions, positions of octarepeat sequence (60-91), and a disulfide bond.

only transmission of spongiform encephalopathies, and their ability to act as protein-only genetic elements.9 Thus, the prion protein offers a considerable challenge to biochemists trying to uncover its precise function and toxicity in cellular milieu, to structural biologists trying to unravel conformational aspects, nucleation, and fibril formation, and to chemists trying to understand coordination geometry, cooperativity of metal binding, and related phenomenon. We have been involved in probing the role of prion octarepeat sequences as potential focal points of oligomerization/aggregation. In this context, we recently reported that peptide conjugates, containing a truncated pentapeptide from the prion octarepeat, exhibit fibrillar structures upon aging, degradation of dopamine, and extensive oxidative damage to plasmid DNA.10 Herein, we wish to report time-dependent growth of fibers in a peptide conjugate 6, containing two identical pentapeptide segments (PHGGG), tethered with a 6-aminocaproic acid (Aca) linker (Scheme 1). Aggregation propensity of this pentapeptide is remarkably accentuated in the bis conjugate resulting in a phased growth of peptide fibrils from spherule-like prefibrillar structures. In addition, it was also observed that known microtubule poison colchicine inhibited that growth of peptide fibers in a concentration-dependent fashion. Ultrastructural details of fiber growth and their inhibition is presented in this paper. Experimental Section General. Dichloromethane, N,N-dimethylformamide, methanol, triethylamine, and 1,2-dimethoxy ethane were distilled

10.1021/jp066546a CCC: $37.00 © 2007 American Chemical Society Published on Web 03/21/2007

Phased Fiber Growth in Peptide Conjugate

J. Phys. Chem. B, Vol. 111, No. 14, 2007 3751

SCHEME 1: Solution-Phase Synthesis of Bis Conjugate 6

following standard procedures prior to use. N,N′-Dicyclohexylcarbodiimide, N-hydroxybenzotriazole, t-butyloxycarbonyl carbonate, trifluoroacetic acid, 6-aminocaproic acid, and L-amino acids were purchased from Spectrochem, Mumbai, India and used without further purification. 1,2-Ethanedithiol and triisopropylsilane were purchased from Lancaster. 1H and 13C NMR spectra were recorded on a JEOL-JNM LAMBDA 400 model operating at 400 and 100 MHz, respectively. Mass spectra were recorded at RSIC, Lucknow, India on a JEOL SX 102/DA6000 mass spectrometer data system using Argon/Xenon (6kV, 10mA) as the FAB gas. Elemental analyses (C, H, N) were performed on a Perkin-Elmer 240-C automatic elemental analyzer. Peptide Conjugate Synthesis. The PHGGG pentapeptide was prepared using a fragment condensation approach following standard solution-phase synthetic protocols (see Supporting Information). Bis conjugate 6 was prepared by tethering two such pentapeptide sequences with 6-aminocaproic acid, followed by purification on a silica gel gravity column (Scheme 1). Purity of 6 was determined by FPLC (>98%), which was further confirmed by FAB mass spectral and CHN analysis. Nr-(tert-Butyloxycarbonyl)-L-prolyl-Nim(trityl)-L-histidylglycyl-glycyl-glycyl-caproic Acid Methyl Ester (2): NR-(tbutyloxycarbonyl)-L-prolyl-Nim(trityl)-L-histidyl-glycyl-glycylglycine (2.3 g, 3.0 mmol), HOBT (0.54 g, 4.0 mmol), was dissolved in dry DMF (10 mL) under nitrogen atmosphere at room temperature. The reaction mixture was cooled to 0 °C under a nitrogen atmosphere, and a solution of DCC (0.82 g, 4.0 mmol) in dry DCM (10 mL) was added dropwise into the reaction mixture. The reaction mixture was stirred for 1 h at 0 °C under nitrogen atmosphere. After 1 h of stirring, 6-amino caproic acid methyl ester hydrochloride (0.67 g, 3.6 mmol) was added in one portion into the cold reaction mixture followed by triethyl amine (0.56 mL, 4.0 mmol). The reaction mixture was stirred for 5 min at cold conditions; then, the reaction

mixture was stirred at room temperature overnight under a nitrogen atmosphere. The reaction mixture was filtered, and the filtrate was concentrated to remove DMF completely; the residue was dissolved in DCM, and the organic layer was washed with 1 N HCl (3 × 20 mL), 10% NaHCO3 (3 × 20 mL), and finally with brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude compound was purified through neutral aluminum oxide (active) column chromatography by using dichloromethane and methanol (97:3) solvent system to give pure compound 3 (1.0 g, 1.1 mmol, yield 37%, mp 158-160 °C, Rf [5% methanol in dichloromethane] 0.6, 1 [R]25 D -37 [c 0.72, methanol]). H NMR (400 MHz, CDCl3, TMS, δ ppm): 1.15 (s, 9H, t-Boc H); 1.25 (m, 2H, linker’s -CH2); 1.44 (m, 2H, linker’s -CH2); 1.53-1.57 (m, 2H, linker’s -CH2); 1.92-2.05 (m, 4H, overlap signals for Pro γ H and linker’s -CH2); 2.24 (m, 2H, Pro β H); 2.94 (m, 1H, His β H); 3.12-3.14 (m, 3H, His β H and linker’s -CH2); 3.40-3.43 (m, 2H, Pro δ H); 3.57 (s, 3H, -OMe); 3.7-4.09 (m, 6H, overlap signals for Gly -CH2); 4.20-4.24 (dd, 1H, J ) 7.5 and 7.0 Hz, Pro R H); 4.53 (m, 1H, His R H); 6.65 (s, 1H, His ring’s H); 6.7 (m, 1H, -NH); 6.95 (br s, 6H, signal for trityl 6H); 7.02 (s, 1H, His ring’s H); 7.34 (m, 10H, overlap signals for trityl H and -NH); 7.66-7.69 (m, 2H, -NH); 9.01 (br s, 1H, -NH). 13C NMR (100 MHz, CDCl3, δ ppm): 24.5, 24.7, 26.2, 27.5, 28.3, 28.9, 29.6, 30.1, 33.8, 39.1, 43.2, 47.2, 51.4, 54.3, 61.9, 75.5, 81.2, 120.5, 128.1, 128.3, 129.6, 136.0, 138.2, 141.8, 156.1, 169.1, 169.8, 170.4, 172.5, 174.1, 174.8. IR (KBr, cm-1): 1524 (amide II); 1633 (amide I); 3294 (-NH str). FAB MS m/z: [M + 1] 893. Anal. Calcd for C48H60N8O9: C, 64.56; H, 6.77; N, 12.55. Found C, 64.85; H, 6.50; N, 11.95. Nr-(tert-Butyloxycarbonyl)-L-prolyl-Nim(trityl)-L-histidylglycyl-glycyl-glycyl-caproic acid (3): Compound 2 (1 g, 1.1 mmol) was dissolved in methanol (6 mL), and 1 N NaOH (1.7 mL) was added to it at room temperature with constant

3752 J. Phys. Chem. B, Vol. 111, No. 14, 2007 stirring. The reaction mixture was stirred for 2 h at room temperature. After this time, methanol was removed in Vacuo, and the residue was cooled to 0 °C, acidified with 1 N HCl, and extracted with dichloromethane. The organic layer was washed with brine and dried over anhydrous sodium sulfate; the solvent was evaporated to give compound 4 (0.95 g, 1.08 mmol, yield 96.9%, mp 138-140 °C, Rf [5% methanol in 1 dichloromethane] 0.3, [R]25 D -29 [c 0.74, methanol]). H NMR (400 MHz, CDCl3, TMS, δ ppm): 1.20-1.32 (m, 4H, linker’s -CH2); 1.39 (s, 9H, t-Boc); 1.42-1.52 (m, 2H, linker’s -CH2); 1.62-1.76 (m, 3H, Pro γ H, β H); 2.04 (br s, 1 H, Pro β H); 2.24 (br s, 2H, linker’s -CH2); 3.15 (br s, 3H, His β H, linker’s -CH2); 3.39 (br s, 3H, Pro δ H, His β H); 3.57-4.15 (m, 6H, overlap signals of Gly -CH2); 4.25 (m, 1H, Pro R H); 4.83 (m, 1H, His R H); 7.12 (br s, 7H, overlap signals for trityl H and His ring’s H); 7.40 (br s, 10H, overlap signals for trityl H and His ring’s H); 7.75 (br s, 1H, -NH); 8.26 (m, 2H, -NH); 8.46 (br s, 2H, -NH). 13C NMR (100 MHz, CDCl3, δ ppm): 24.0, 24.5, 25.6, 28.3, 29.6, 30.3, 31.5, 33.8, 38.8, 43.1, 43.8, 47.3, 52.4, 60.8, 78.7, 80.6, 121.9, 128.7, 129.1, 129.5, 131.5, 135.0, 139.6, 155.0, 169.8, 170.8, 176.1. FAB MS m/z: [M + 1] 879. Anal. Calcd for C47H58N8O9: C, 64.22; H, 6.65; N, 12.75. Found: C, 63.95; H, 6.80; N, 12.65. Nr-(tert-Butyloxycarbonyl)-L-prolyl-Nim(trityl)-L-histidylglycyl-glycyl-glycyl-caproyl-L-prolyl-Nim(trityl)-L-histidyl-glycyl-glycyl-glycine Methyl Ester (4): Compound 3 (0.1 g, 0.1 mmol), HOBT (0.018 g, 0.13 mmol), were dissolved in dry DMF (0.5 mL) under a nitrogen atmosphere, and the reaction mixture was cooled to 0 °C. A solution of DCC (0.028 g, 0.13 mmol) in dry dichloromethane was added into the reaction mixture dropwise at 0 °C. The reaction mixture was stirred for 1 h at 0 °C. After 1 h, a solution of l-prolyl-Nim(trityl)-L-histidylglycyl-glycyl-glycine methyl ester (intermediate X) in dichloromethane (2 mL) was added in one portion into the reaction mixture at 0 °C. The reaction mixture was stirred for 5 min at 0 °C, stirred for 18 h at room temperature, and then filtered in suction. Filtrate was concentrated to remove DMF completely. The residue was dissolved in dichloromethane. The organic layer was washed with 1 N HCl (3 × 15 mL), 10% NaHCO3 (3 × 15 mL), and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated. The crude compound was purified through neutral aluminum oxide (active) column chromatography by using a dichloromethane and methanol (96: 4) solvent system to give pure compound 5 (100 mg, 0.064 mmol, yield 57%, mp 140-142 °C, Rf [6% methanol in 1 dichloromethane] 0.4, [R]25 D -38 [c 0.60, methanol]). H NMR (400 MHz, CDCl3, TMS, δ ppm): 1.2-1.25 (m, 11H, overlap signals for t-Boc and linker’s -CH2); 1.34-1.40 (m, 4H, linker’s -CH2); 2.00-2.05 (m, 6H, Pro γ H, linker’s -CH2); 2.29-2.30 (m, 4H, Pro β H); 2.94 (m, 2H, His β H); 3.103.14 (m, 4H, His β H, linker’s -CH2); 3.51-3.55 (m, 4H, Pro δ H); 3.66 (s, 3H, -OMe); 3.79-4.30 (m, 12H, overlap signals for Gly -CH2); 4.4-4.6 (m, 4H, Pro R H, His R H); 6.636.66 (m, 2H, overlap two singlet, His ring’s H); 6.8 (m, 1H, -NH); 7.03-7.20 (m, 13H, overlap signals for -NH and trityl H); 7.25 (br s, 2H, His ring’s H); 7.31-7.35 (br s, 18H, trityl H); 7.4 (m, 1H, -NH), 7.62 (m, 2H, -NH); 7.8 (m, 2H, -NH); 8.7 (br s, 1H, -NH); 9.0 (br s, 1H, -NH). 13C NMR (100 MHz, CDCl3, δ ppm): 23.8, 24.7, 24.9, 26.1, 27.7, 28.3, 28.7, 29.4, 34.5, 38.9, 40.8, 42.8, 43.0, 43.3, 47.2, 47.8, 52.0, 54.2, 54.9, 61.7, 75.4, 75.5, 81.3, 120.0, 120.5, 128.16, 128.19, 128.34, 128.39, 129.54, 129.62, 135.9, 138.3, 141.8, 141.9, 154.0, 169.1, 169.8, 170.2, 170.5, 173.9. IR (KBr, cm-1): 1541 (amide II), 1660 (amide I), 3320 (-NH str). FAB MS m/z: [M + 1] 1541.

Ghosh and Verma Anal. Calcd for C84H97N15O14: C, 65.48; H, 6.35; N, 13.64. Found: C, 65.15; H, 6.80; N, 13.95. L-Prolyl-Nim(trityl)-L-histidyl-glycyl-glycyl-glycine Methyl Ester (X): NR-(tert-Butyloxycarbonyl)-L-prolyl-Nim(trityl)-Lhistidyl-glycyl-glycyl-glycine methyl ester (0.25 g, 0.31 mmol) was taken in a round-bottom flask and cooled to 0 °C in ice bath. Then, 2 mL of 1 N HCl in 90% acetic acid was added dropwise into the solid. The reaction mixture was stirred for 2 h with slow elevation of temperature. After 2 h, the reaction mixture was slowly poured into the ice cold 10% NaHCO3 solution (20 mL). The aqueous layer was extracted with dichloromethane in cold conditions three times (3 × 20 mL). The combined organic layer was washed with brine and dried over anhydrous sodium sulfate. Again, the dry organic layer was passed through an anhydrous sodium sulfate bed and stored in an ice cold bath under nitrogen atmosphere. The cold organic layer was concentrated to 2 mL from 60 mL in vacuum without heating. Then, a 2 mL solution of intermediate X was immediately used for further synthetic steps (Rf (10% methanol in dichloromethane) 0.4). Nr-(tert-Butyloxycarbonyl)-L-prolyl-Nim(trityl)-L-histidylglycyl-glycyl-glycyl-caproyl-L-prolyl-Nim(trityl)-L-histidyl-glycyl-glycyl-glycine (5): Compound 4 (0.35 g, 0.22 mmol) was dissolved in methanol (3 mL), and 1 N NaOH (0.65 mL) was added to the solution at room temperature with constant stirring. The reaction mixture was stirred for 2 h at room temperature. After 2 h, the methanol was removed in vacuum and the residue was cooled to 0 °C, acidified with 1 N HCl, and extracted with dichloromethane. The organic layer was washed with brine and dried over anhydrous sodium sulfate; the solvent was evaporated to give compound 6 (0.33 g, 0.21 mmol, yield 97%, mp 160162 °C, Rf [1% methanol in dichloromethane] 0.3, [R]25 D -23 [c 0.71, methanol]). 1H NMR (400 MHz, CD3OD, TMS, δ ppm): 1.25-1.27 (m, 4H, linker’s -CH2); 1.35-1.36 (s, 9H, t-Boc); 1.4-1.55 (m, 2H, linker’s -CH2); 1.8-2.04 (m, 4H, Pro γ H); 2.15 (m, 2H, linker’s -CH2); 2.33 (m, 4H, Pro β H); 3.11 (m, 2H, His β H); 3.29-3.30 (m, 4H, His β H, linker’s -CH2 and overlap CD3OD signal); 3.50-3.60 (m, 2H, Pro δ H); 3.78-4.09(m, 12H, overlap signals of Gly -CH2); 4.28 (m, 2H, Pro R H); 4.74 (m, 2H, His R H); 7.19 (br s, 14H, overlap signals for trityl H and His ring’s H); 7.42 (br s, 20H, overlap signals for trityl H and His ring’s H). 13C NMR (100 MHz, CD3OD, δ ppm): 23.7, 25.3, 25.6, 25.9, 26.0, 27.3, 27.6, 28.7, 30.06, 30.4, 30.7, 31.4, 35.2, 40.2, 43.4, 43.8, 53.4, 61.5, 61.7, 66.9, 69.0, 81.5, 88.2, 119.0, 128.0, 128.7, 129.8, 130.1, 130.9, 135.0, 141.5, 145.3, 156.0, 171.9, 172.4, 175.0. IR (KBr, cm-1): 1544 (amide II), 1662 (amide I), 3439 (-NH str). FAB MS m/z: [M + 1] 1526. Anal. Calcd for C83H95N15O14: C, 65.30; H, 6.27; N, 13.76. Found: C, 65.15; H, 5.95; N, 13.85. L-Prolyl-L-histidyl-glycyl-glycyl-glycyl-caproyl-L-prolyl-Lhistidyl-glycyl-glycyl-glycine (6): Compound 5 (0.33 g, 0.21 mmol) was mixed with a cocktail solution (2.5 mL) (composition of 3 mL cocktail solution: 2.7 mL trifluoro acetic acid + 0.15 mL dichloromethane + 0.09 mL EDT + 0.06 mL triisopropylsilane) under nitrogen atmosphere at room temperature. The reaction mixture was stirred for 4 h at room temperature under nitrogen atmosphere. After 4 h, the solvent was evaporated and the solid residue was washed with diethyl ether (4 × 15 mL) and dichloromethane (2 × 15 mL). The solid was dissolved in a water/methanol (1:1) mixture and passed through a strong anion exchange resin. Water and methanol were removed, and the solid was dissolved in methanol again to be precipitated out by dropwise addition of diethyl ether. This process was repeated three times. Finally, the compound was

Phased Fiber Growth in Peptide Conjugate dried under high vacuum to give compound 6 (0.1 g, 0.1 mmol, yield 50%, mp could not be checked (too hygroscopic), Rf [n-Butanol/acetic acid/water; 1/0.25/0.42] 0.4, [R]25 D -57 (c 0.82, methanol). 1H NMR (400 MHz, D2O, TMS, δ ppm): 1.16 (m, 2H, linker’s -CH2); 1.38-1.46 (m, 4H linker’s -CH2); 1.55 (m, 2H, Pro γ H); 1.67-2.08 (m, 5H, overlap signals for Pro γ H, linker’s -CH2 and Pro β H); 2.26 (m, 3H, overlap signals for Pro 1β H and Pro 2β H); 2.69-2.75 (m, 1H, Pro δ H); 2.90-3.05 (m, 4H, overlap signals for His β H and linker’s -CH2); 3.21 (m, 1H, Pro δ H); 3.47 (m, 1H, Pro δ H); 3.603.63 (3H, overlap signals of broad singlet and multiplate for Gly -CH2 and Pro δ H); 3.76 (br s, 2H, Gly -CH2); 3.813.84 (m, 8H, overlap signals for Gly -CH2); 3.99-4.04 (dd, J ) 8.4 and 8.0 Hz, 1H, Pro R H); 4.21 (m, 1H, Pro R H); 4.484.65 (m, 2H, His R H); 6.85 (s, 2H, His ring’s H); 7.55 (s, 2H, His ring’s H). 13C NMR (100 MHz, D2O, δ ppm): 24.5, 25.0, 25.9, 26.4, 28.8, 30.2, 31.1, 34.6, 39.9, 43.1, 43.2, 43.3, 43.4, 43.6, 43.9, 47.9, 48.8, 54.4, 61.1, 62.1, 117.8, 134.5, 136.9, 171.7, 172.6, 175.4, 177.1, 178.5. IR (KBr, cm-1): 1594 (amide II), 1656 (amide I), 3433 (-NH str). FAB MS m/z: [M + 1] 942. Anal. Calcd for C40H59N15O12: C, 51.00; H, 6.31; N, 22.30. Found: C, 51.35; H, 6.67; N, 22.75. Analytical HPLC Purity: >98% (see Supporting Information). CD Spectroscopy. CD spectra were recorded at 25 °C under a constant flow of nitrogen on a JASCO-810 spectropolarimeter, which was calibrated with an aqueous solution of ammonium D-(+)-camphor sulfonate. Experimental measurements were carried out in water or at different pH values in 5 mM phosphate buffered solution (sample concentration was 0.33 mM) by using a 1 mm path length cuvette between 190-400 nm. The spectrum represents an average of 5-8 scans, and the CD intensities are expressed in mean-residue ellipticity. Origin 6.0 professional software and adjacent averaging for the smoothing function were used to process CD data. Optical Microscopy (OM). Congo red dye (3 µM in 100 mM NaCl) was added to aged solutions of 6 (0.33 mM) and incubated for 6 h at room temperature. Then, 50 µL of this solution was transferred onto a glass slide, dried, and then observed under an optical microscope (Zeiss AX100) under cross-polarized light. Images were processed by using ImagePro Plus. For time-course imaging, aliquots of aged peptide were drawn during the prolonged incubation. Atomic Force Microscopy (AFM). Fresh and aged peptide samples were imaged with an atomic force microscope (Molecular Imaging) operating under Acoustic AC mode (AAC), with the aid of cantilever (NSC 12(c) from MikroMasch). The force constant was 0.6 N/m; the resonant frequency was 150 kHz. The images were taken in air at room temperature with the scan speed of 1.5-2.2 lines/sec. The data acquisition was done using PicoScan 5 software; the data analysis was done with the aid of visual SPM. Conjugate 6 (0.33 mM) was incubated for 0-31 days in water, and micrographs were recorded for selected incubation periods. Then, 10 µL of aqueous solution of 6 was transferred onto a freshly cleaved mica surface and uniformly spread with the aid of a spin-coater operating at 200-500 rpm (PRS-4000). The sample-coated mica was dried for 30 min at room temperature, followed by AFM imaging. Scanning Electron Microscopy (SEM). SEM measurements were performed on a FEI QUANTA 200 Microscope equipped with a tungsten filament gun. The micrograph for 6 was recorded at WD 10.6 mm, magnification 40000×. The concentration of peptide samples or metal complexes used was 0.33 mM.

J. Phys. Chem. B, Vol. 111, No. 14, 2007 3753 Results and Discussion Time-Dependent Aggregation of Peptide Conjugate 6. Aca has been used to connect two peptide segments or as a bifunctional linker in the synthesis of cyclic peptides.11 Such studies have primarily aimed to bring two peptide geometries in close proximity or for the incorporation of β-turns in cyclic systems. Crystallographic studies indicate that the polymethylene unit of Aca can adopt various conformations ranging from a zigzag orientation to a helical pattern and also offers ample flexibility to peptide backbones to achieve an extended conformation.12 Synthesis of conjugate 6 employed solution-phase chemistry, followed by spectroscopic analyses to confirm its purity (see Supporting Information). We performed microscopic studies within the concentration range of 0.1-1 mM and observed a distinct effect of concentration on peptide aggregation. At lower concentrations, peptide fibers were either ill-formed or sparsely distributed; higher concentrations lead to dense fibrillation. Thus, we decided to use 0.33 mM concentration of 6 for aggregation studies in pure water. The samples were aged at 37 °C for 0-30 days, and aliquots were occasionally drawn to follow the timedependent self-assembly process by the help of microscopic detection. Appearance of Spherical Structures in the Initial Phase of Aggregation. We visualized time-dependent aging of 6 by observing spherical structures, stained with Congo red dye, at day 1 under a dark-field polarizing microscope. The growth of fibrous patterns from spherical cores on days 3 and 7 was evident in the early phase of fiber growth, which culminated into long fibrils at the end of day 15 and beyond (Figure 2). Early phases of aging afforded an insight into sequential growth of the fibers from a disorganized core of spherulite-like structure to the emergence of thick fibrils out of the circular core, finally to its metamorphosis into fine tubular structure with complete disappearance of prefibrillar spherulites at day 15. It is worth mentioning that all the microscopic images reported were reproducible and the prefibrillar structures generated were detected in several samples aged in parallel. Similar gross structural morphologies have been observed for several proteins such as variable domain SMA of immunoglobulin light chains, amyloid-β, silkmoth chorion protein, and R-synuclein, to name a few.13 Such spherical morphologies reflect long-term ordering of proteinaceous structures and are suggestive of actively evolving intermediates in the course of progressive growth of full-length mature protein/peptide fibers.14 These observations have a parallel in material science where spherical (spherulite) precursors are invoked for the growth of crystalline materials.15 Interestingly, the PHGGG pentapeptide alone did not exhibit extensive aggregation, but afforded formation of ill-defined fibers upon prolonged incubation period. Ultrastructural Details Probed by Microscopic Analysis. Formation of spherical morphologies in fresh solution to elongated fibers upon aging in 6 suggested a definite propensity of this conjugate to rapidly self-assemble in aqueous solution. We resorted to AFM to further probe the nature of spherical and fibrous structures. Aged solutions of 6 were subjected to AFM analysis at various incubation time points, including a freshly prepared solution. Akin to the observation of spherical prefibrillar structures in optical imaging, AFM micrographs also revealed the presence of punctuated, spherical structures in a freshly prepared solution of 6, which eventually culminated into discrete fibrous patterns upon aging for 15 days (Figure 3). Many fibers displayed a persistent length of 50 µm with a cross-

3754 J. Phys. Chem. B, Vol. 111, No. 14, 2007

Figure 2. (a) Snapshots of time-dependent growth of peptide conjugate 6 in water. Spherical prefibrillar structures are evident at day 1, followed by fibrous growth from spherical nuclei (day 3); extension of fibrils (day 7); and, finally, formation of fully extended fibrils (day 15). (b) White arrow indicates outer shell of the double-layered spherical prefibrillar structure; red arrow indicates the core stained by Congo red.

sectional diameter of ∼2 µm, thus indicating an ordered growth of fibers upon longer incubation times. Discrete prefibrillar spherical structures imaged by force microscopy further confirm their occurrence and possible role in elongation and growth of fibrillar structures. It is likely that these spherical intermediates play a crucial role in nuclei formation necessary for the early phase(s) of fiber growth. Prolonged incubation of 30 days afforded dense fibrillar structures, and the internal diameter of the peptide fibers narrowed down to the nanometric dimensions (20-40 nm) (Figure 4). Mixed solvent regime afforded a change in the morphology of peptide fibers derived from 6. A mixture of aqueous trifluoroethanol (TFE) was employed instead of pure water to study aggregative propensity and ensuing morphology of fibrous patterns generated upon aging. Incubation of 6 in 60% TFEwater resulted in super-aggregation of fibers into fibrous bundles as evidenced in flattened and derivative AFM micrographs (Figure 5). Moreover, peptide fibril bundles arranged themselves in a distinct braided, twisted rope-like morphology, as verified by SEM analysis, when incubated in 80% TFE-water mixture for 15 days (Figure 6). This observation further confirmed fibrous bundle formation and suggested that higher TFE

Ghosh and Verma

Figure 3. AFM micrographs of 6. (a) Image confirms the presence of punctuated, spherical structures in the fresh sample (see inset). (b) Existence of persistent length elongated fibers in 15 days aged solution of 6. Cross-sectional analysis revealed 2 µm internal diameter of peptide fibers.

concentration was able to coerce peptide fibers to stick together. This example clearly shows a remarkable effect of solvent in modulating gross morphology and the aggregative pattern of peptide conjugates upon aging. CD Spectral Analysis of 6 in TFE-Water Mixture. We decided to study the possibility of induced secondary structural feature in 6 by CD analysis to ascertain the effect of TFE addition in aqueous medium. The motivation for this study comes from the fact that the polymethylene unit of Aca, present in conjugate 6, can adopt various conformations ranging from a zigzag orientation to a helical pattern12 and that TFE is a solvent known to influence stability of peptide secondary structures, with a distinct inclination for R-helical features.16 Interestingly, induction and stabilization of R-helicity was also observed upon incubation of 6 in TFE-water medium (Figure 7). The fresh sample as well as the 15 day aged solution demonstrated persistent appearance of signals corresponding to R-helices, thus suggesting that the fibers so formed upon aggregation contain helicity, unlike the more commonly attributed β-sheet character for aggregating peptide and proteins.

Phased Fiber Growth in Peptide Conjugate

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Figure 4. AFM micrograph revealing dense peptide fibrous structures from 6 after 30 days of incubation.

Figure 6. SEM micrographs of 15 day aged solution of 6 in 80% TFE-water. (a) Thick peptide fiber and (b) fibrous bundles with a braided morphology (scale bar corresponds to 2 µm). Figure 5. (a) Flattened and (b) derivative AFM micrographs revealing fibrous bundles in a 15 day aged solution of 6 in 60% TFE-water.

TFE can affect secondary structures via a combination of hydrogen bonding, hydrophobic interactions, and clustering of alcohol molecules.17 However, a detailed understanding of preferential stabilization of secondary structures by fluorinated alcohols remains elusive. In our case, it is reasonable to assume that nonpolar polymethylene chains of Aca and six glycine residues in 6 are considerably desolvated by TFE, affording a helical conformation to maximize the hydrophobic effect. Thus,

a transition from the random coil behavior of 6 in pure water to helical structure in increasing amount of TFE is observed with the addition of TFE. Such a desolvation model has been previously documented for an alanine-rich peptide (AAKAA)n, where the helical state was found to be stabilized to a greater extent compared to its random coil state.17a Incidentally, R-helical structures also exhibit the propensity to aggregate as demonstrated for the tau and Ure2p protein.18 It was proposed that the aggregation process involved arrangement of R-helices into coiled coils. Therefore, it is not surprising

3756 J. Phys. Chem. B, Vol. 111, No. 14, 2007

Figure 7. CD traces of fresh and 15 days aged solution of 6 in pure water and 80% TFE-water.

Figure 8. CD spectra of colchicine (0.1 mM) alone and with 6 (0.33 mM), in water and phosphate buffer (PB) at pH 7.0.

that despite structural transition in 6 going from water to TFEwater mixtures there is a definite change in overall conformation as proved by the CD studies, but its ability to aggregate remains unaltered. It is likely that small models can provide interesting insight into conformational polymorphisms between R-helices, β-sheets, polyproline helices, and a host of other conformations. It is emphasized that CD spectra of aggregating peptides may

Ghosh and Verma contain errors due to the presence of insoluble aggregates, so caution must be exercised in interpreting such spectral results. Colchicine Mediated Disaggregation. Having demonstrated time-dependent aggregation in 6, a truncated construct from prion octarepeats, we focused our attention toward the possibility of using this system to screen for inhibitors of peptide/protein aggregation. As a first step toward this goal, we decided to probe the effect of colchicine on the aged solutions of conjugate 6. Colchicine is a known antimitotic agent that exerts its mechanism of action by inhibiting microtubule assemblies,19 and interestingly, it has also been found to interact with systemic amyloidosis conditions.20 The interaction of colchicine with 6 was first investigated by adding this alkaloid to a solution of 6, followed by CD spectral analysis. In the case of a colchicine-tubulin interaction, a decrease in the intensity of negative ellipticity band at 340 nm, characteristic of the methoxytropone ring system, is suggestive of a favorable drug-protein interaction.21 This diagnostic indicator was also used to analyze interaction between the colchicine-peptide conjugate, where a discernible reduction in negative ellipticity at 340 nm in water and phosphate buffer (5 mM, pH 7.0) suggested possible binding of alkaloid to 6 in solution (Figure 8). Encouraged by this observation, we decided to study the effect of colchicine on time-dependent aggregation of 6. The premise of colchicine-6 binding was extended to aged solutions to examine whether this interaction will lead to disaggregation of peptide fibrils. In this connection, we either co-aged colchicine (0.5 mM) together with 6 from day 0 or it was added to a 10 day aged solution of 6. In the former case, co-incubation completely inhibited peptide fibril formation as analyzed by optical and force microscopy (Figure 9a,b). This sequence of addition resulted mostly in the formation of punctated spherical structures, without any trace of fibrillation. In another experiment, colchicine was added to a 10 days aged solution of 6, followed by an incubation of 7 days. This led to a different morphological landscape where punctated structures as well as short dissolved fibrils were observed when subjected to microscopic evaluation (Figure 9c). These results suggest that short fibrillating peptides can be used as a viable screen to identify inhibitors of aggregation or facilitators of aggregate dissolution.4a However, the precise mechanism of the colchicine-6 interaction remains unclear at the present time and is being elucidated. Conclusions A bis pentapeptide conjugate 6 was synthesized by solutionphase methodology and its aggregation behavior studied by microscopic analysis. Phased growth of peptide fibers in

Figure 9. (a) Optical and (b) atomic force micrographs of co-incubation of colchicine and 6. (b) AFM micrograph of colchicine addition to 10 day aged solution of 6.

Phased Fiber Growth in Peptide Conjugate conjugate 6, derived from a prion octarepeat sequence, offers an interesting and intriguing insight into the aggregation pathway with sequential occurrence of peptide oligomerization intermediates. The reproducible appearance of prefibrillar spherulitic structures point toward the fact that aggregation of short peptide sequences is a culmination of various conformational ensembles possessing discreet structures. The mechanism of fibrillation may involve interaction of 6 through backbone interactions in an extended orientation as suggested for certain Aca constructs. Bis conjugation using the Aca linker allows for a higher number of hydrogen bonding interactions compared to a simple dipeptide, and this increased stabilization by hydrogen bonding may lead to the formation of peptide fibers. However, this inference based on CD and microscopic evidence is speculative in nature, and a more thorough treatment of structure-fibrillation propensity of peptide conjugate 6 is only possible with the help of high-resolution NMR studies to identify a preponderance of structural motif in 6 and their role in fiber formation. Moreover, we were also able to demonstrate that the antimitotic agent colchicine afforded disaggregation of aged 6 solutions thus suggesting it as a suitable model to screen for novel inhibitors of aggregation. We aim to further investigate application of short peptide sequences for the detection of fibrillation pathways by solution-phase and solid-state NMR techniques. Studies directed toward these possibilities are currently under active consideration. Acknowledgment. We would like to thank Professors A. Sharma, Chemical Engineering, and P. Sinha, Biological Sciences and Bioengineering, IIT-Kanpur, for access to the AFM machine. Other microscopic investigations were carried out in ACMS, IIT-Kanpur. S.G. thanks IIT-Kanpur for a pre-doctoral fellowship, and S.V. thanks the Department of Science and Technology, India, for financial support through a Swarnajayanti Fellowship in Chemical Sciences. An anonymous referee is gratefully acknowledged for insightful comments on CD studies in the presence of TFE and colchicine. Supporting Information Available: Solution phase synthesis, HPLC chromatogram, and optical images of 6. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) (a) Ross, C. A.; Poirier, M. A. Nature ReV. Mol. Cell Biol. 2005, 6, 891-898. (b) Ross, C. A.; Poirier, M. A. Nat. Med. 2004, S10-S17. (c) Caughey, B.; Lansbury, P. T., Jr. Ann. ReV. Neurosci. 2003, 26, 267-298. (d) Soto, C. Nature ReV. Neurosci. 2003, 4, 49-60. (d) Dobson, C. M. Philos. Trans. R. Soc. London, Ser. A 2001, 356, 133-145. (2) (a) Tabner, B. J.; El-Agnaf, O. M. A.; German, M. J.; Fullwood, N. J.; Allsop, D. Biochem. Soc. Trans. 2005, 33, 1082-1086. (b) Stefani, M. Biochim. Biophys. Acta 2004, 1739, 5-25. (b) Dobson, C. M. Nature 2003, 426, 884-890. (3) (a) Petty, S. A.; Adalsteinsson, T.; Decatur, S. M. Biochemistry 2005, 44, 4720-4726. (b) Dou, Y.; Baisnee, P.-F.; Pollastri, G.; Pecout, Y.; Nowick, J. S.; Baldi, P. Bioinformatics 2004, 20, 2767-2777. (c) Chiti, F.; Taddei, N.; Bucciantini, M.; White, P.; Ramponi, G.; Dobson, C. M. EMBO J. 2000, 19, 1441-1449. (4) (a) Gazit, E. FEBS J. 2005, 272, 5971-5978. (b) Ventura, S.; Zurdo, J.; Narayanan, S.; Parreno, M.; Mangues, R.; Reif, B.; Chiti, F.; Giannoni, E.; Dobson, C. M.; Aviles, F. X.; Serrano, L. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 7258-7263. (c) Ivanova, M. I.; Sawaya, M. R.; Gingery, M.; Attinger, A.; Eisenberg, D. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 1058410589. (d) Reches, M.; Gazit, E. Science 2003, 300, 625-627. (5) (a) Liebman, S. W. Nature Struct. Mol. Biol. 2005, 12, 567-568. (b) Nelson, R.; Sawaya, M. R.; Balbirnie, M.; Madsen, A. O.; Riekel, C.; Grothe, R.; Eisenberg, D. Nature 2005, 435, 773-778. (c) Krishnan, R.; Lindquist, S. L. Nature 2005, 435, 765-772. (d) Ritter, C.; Maddelein, M.-L.; Siemer, A. B.; Luehrs, T.; Ernst, M.; Meier, B. H.; Saupe, S. J.; Riek, R. Nature 2005, 435, 844-848.

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