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Formation of Annular Protofibrillar Assembly by Cysteine Tripeptide: Unravelling the Interactions with NMR, FTIR and Molecular Dynamics Biswadip Banerji, Moumita Chatterjee, Uttam Pal, and Nakul Chandra Maiti J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b04373 • Publication Date (Web): 08 Jun 2017 Downloaded from http://pubs.acs.org on June 15, 2017

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The Journal of Physical Chemistry

Formation of Annular Protofibrillar Assembly by Cysteine Tripeptide: Unravelling the Interactions with NMR, FTIR and Molecular Dynamics

Biswadip Banerjia,* Moumita Chatterjee a, Uttam Pal,b and Nakul C. Maitib,*

a

Organic and Medicinal Chemistry, CSIR-Indian Institute of Chemical Biology; 4, Raja S.C.

Mullick Road, Kolkata 700032, West Bengal, India. Fax: +91-33-2473-5197; Tel: +91-332499-5709; E-mail: [email protected]

b

Structural Biology & Bio-informatics Division, CSIR-Indian Institute of Chemical Biology;

4, Raja S.C. Mullick Road, Kolkata 700032, West Bengal, India. Fax: +91-33-2473-5197; Tel: +91-33-2499-5940; E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT Both the hydrogen bonding and hydrophobic interactions play a significant role in molecular assembly including self assembly of proteins and peptides. In this investigation, we reported formation of annular protofibrillar structure (diameter ~500 nm) made of a newly synthesized s-benzyl-protected cysteine tripeptide which was primarily stabilized by hydrogen bonding and hydrophobic interactions. AFM and FESEM analyses found small oligomers (~ 60 nm in diameter) to bigger annular (~ 300 nm) with an inner diameter of 100 nm and protofibrillar structure after 1-2 days incubation. ROESY NMR spectral analysis revealed the presence of several non-bonded proton-proton interactions among the residues; such as amide protons with methylene group, aromatic protons with tertiary butyl group, methylene protons with the tertiary butyl group etc. These added significant stabilization to bring the peptides closer to form a well ordered assembled structure. H/D exchange NMR measurement further suggested that two individual amide protons among the three amide groups were strongly engaged with the adjacent tripeptide via H-bond interaction. However, the remaining amide proton was found to be exposed to solvent and remained non-interacting with the other tripeptide molecules. In addition to chemical shift values, a significant change in amide bond vibrations of the tripeptide was found due to self assembly formation. The amide I mode of vibrations involving two amide linkages appeared at 1641 cm−1 and 1695 cm−1 in the solid state. However, in the assembled state the stretching band at 1695 cm-1 became broad and slightly shifted to ~1689 cm-1. On the contrary, the band at 1641 cm−1 shifted to 1659 cm-1 and indicated that the -C=O bond associated with this vibration became stronger in the assembled state. These changes in FTIR frequency clearly indicated changes in amide backbone conformation and associated hydrogen bonding pattern due to formation of the assembled structure. In addition to hydrogen bonding, molecular dynamics simulation indicated that the number of π-π interactions also increased with increasing the number of tripeptide participated in the self assembly process. Combined results envisaged a cross β-sheet assembly unit consisted of four intermolecular hydrogen bonds. Such non-covalent peptide assemblies glued by hydrogen bonding and other weak forces may be useful in developing nano-capsule and related materials.


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INTRODUCTION Molecular self-assembly1 is the process of spontaneous organization of the disordered molecules to well defined structures.2,3 The assembly formation of molecules is not just confined to a particular group of molecules, it spans from small inorganic complexes4 to bio macromolecules such as proteins5,6 and DNA7. Several amyloidogenic proteins8 are found to self assemble and produce fibrillar structure and in the process they also produce different intermediate structures like annular oligomers and diffusible protofibrillar structure.9,10 Apart from the amyloidogenic proteins, generation of fibrillar and protofribillar structure through self-assembly of small peptides11-13 has also been reported in recent years. This aggregation of amyloidogenic proteins and peptides is facilitated through self assembly phenomenon. For instance amyloid beta peptides (Aβs) form several assembled structures with different morphologies and these species, particularly oligomeric structures are very much implicated in the pathology of Alzheimer’s disease (AD).14 Oligomer/annular protofibril generation is an intermediate step15 of amyloid-β fibrillogenesis associated with the AD. Being ring-shaped or pore-like structures, annular protofibrils/oligomers may disrupt the cell membrane easily.16,17 It is, therefore, necessary to understand the molecular mechanism behind the assembly process to arrest fibrillar and protofibrillar assembly generation.

However the transformation of the peptides and proteins from their normal soluble state to fibrillar state is a complex process and the molecular detail of the assembled structures are not very well known. The formation of molecular assembly of the peptides and proteins and their stabilization may be governed by both the intramolecular and intermolecular interactions18 including van der Waals, electrostatic, hydrogen bonding19 and stacking20 interactions among the engaged molecules. Therefore, various molecular forces exerted the directionality and specific orientation required for molecular assembly generation by the peptides for developing diverse nanostructures.21

Here in this article, we showed molecular details of the assembled structure, particularly annular protofibrillar assembly formed by novel cysteine tripeptide. This tripeptide is Nterminally protected by tert-butoxycarbonyl (BOC) group and C-terminally protected by methyl ester group. In addition, the side chain of each cysteine residue is protected by benzyl group. It has been reported that the s-benzyl protected cysteine dipeptide forms nanotubes in solution phase.22 It seems that π-π stacking offered by the side chain benzyl group and the



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intermolecular hydrogen bonding through the amide linkage, acid along with amine group provided the orientation for nanotube formation by the s-benzyl protected cysteine dipeptide. We purposefully synthesized this s-benzyl-protected cysteine tripeptide to determine whether the incorporation of the third s-benzyl protected cysteine, amide linkage and terminal protecting group into the dipeptide analogue, could alter the triggering force for molecular assembly and affect the ultimate self assembled structure compared to its dipeptide analogue. Consequently, various microscopy image analyses have been employed to characterize the morphology of the assembled structure generated by this tripeptide. In order to find out the molecular forces responsible for this tripeptide assembly, NMR, FTIR along with molecular dynamics analyses have been performed. In addition, CD spectroscopy, UV-vis spectroscopy established the self assembly phenomenon associated with this tripeptide. Vibrational spectroscopy23 including infrared spectroscopy (IR) is the most significant tool for elucidating the protein/peptide structural aspect.24,25 The bands appeared in FTIR26 are correlated to the molecular arrangement of the peptide backbone by analyzing the most sensitive band like amide I, amide II and amide III which are mainly originating from backbone vibrations. Herein, FTIR spectra investigate the structural orientation of this tripeptide in solid, monomeric and aggregated state. 2D NMR especially ROESY spectra is useful for characterizing the through space interactions present in the peptide. In this context, presence of various non bonded proton-proton interactions and the hydrogen bonding interactions in the aggregated state of the tripeptide have been elucidated by ROESY spectra. The molecular interactions as obtained from the molecular dynamics analysis corroborate the result of NMR spectral analysis. CD spectroscopy validated the self assembly affinity of this tripeptide and hence authenticates other experimental results. MATERIALS AND METHODS Reagents: All chemicals were purchased from acros organics and used without further purification unless otherwise stated. Solvents were freshly distilled by the standard procedures prior to use. Column chromatography was performed on silica gel (Merck, 60-120 mesh) with the required eluant. Mass spectra were obtained on a Jeol MS station 700. All 1H and 13C-NMR spectra were recorded on Bruker 600 and 300 MHz spectrometer. All FT-IR are recorded on Bruker TENSOR27 spectrometer.


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Procedure for synthesis of the tripeptide: To a well stirred solution of N-(tert-Butoxycarbonyl)-S-benzyl-L-cysteine (1; 480 mg, 1.6 mmol)

dissolved

in

N,N-dimethylformamide

(8

mL),

was

added

anhydrous

hydroxybenzotriazole (HOBT; 300 mg, 1.92 mmol) slowly followed by 1-ethyl-3,3(dimethylamino) propyl carbodiimide hydrochloride (EDC·HCl; 600 mg, 3.2 mmol) in cooled condition under nitrogen atmosphere. Then the stirring was continued for 10 minutes at ice-cooled condition and to this mixture triethylamine (TEA; 1 mL, 7.5 mmol) was added along with S-benzyl-L-cysteine methyl ester (2; 400 mg, 1.6 mmol), subsequently the reaction was further continued for 8 hrs at room temperature (monitoring via TLC). The reaction mixture was then concentrated to dryness and extracted with ethyl acetate (3 × 20 mL) from aqueous layer. Evaporation of solvent left the crude product, which was purified by column chromatography over silica gel (hexane/ethyl acetate 75:25) to afford the intermediate compound‘3’ as white solid (yield = 70%). After that, deprotection of the N(tert-Butoxycarbonyl) group from the intermediate ‘3’ was mediated by 4(M) HCl in 1,4 dioxane to afford the intermediate ‘4’. Evaporation of solvent and azeotrope with the toluene provide the crude product which was directly used to the next step reaction. To carry out the final reaction, anhydrous hydroxybenzotriazole (HOBT; 110 mg, 0.78 mmol) was added followed by the addition of 1-ethyl-3,3-(dimethylamino) propyl carbodiimide hydrochloride (EDC·HCl; 270 mg, 1.4 mmol) to a well stirred solution of N-(tert-Butoxycarbonyl)-Sbenzyl-L-cysteine (1; 172 mg, 0.65 mmol) dissolved in N,N-dimethylformamide at cooled condition under nitrogen atmosphere. After stirring the solution for 10 minutes at ice-cooled condition triethylamine (TEA; 1 mL, 2.25 mmol) was added along with the addition of intermediate ‘4’ (270 mg, 0.65 mmol), then the reaction was further continued for 8 hrs at room temperature (monitoring via thin-layer chromatography (TLC)). The reaction mixture was evaporated to dryness and extracted with ethyl acetate (3 × 20 mL) from aqueous layer. Evaporation of solvent left the crude product, which was further purified by column chromatography over silica gel (hexane/ethyl acetate 60:40) to afford the desired compound ‘5’ (yield = 55%) as white solid (scheme 1).



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Scheme 1. Schematic of synthetic route of the tripeptide. Reagents and Condition (a) EDC·HCl, HOBt, TEA, 0°C to r.t, 8 hrs, (b) 4 M HCl in 1,4-dioxane, 0°C to rt, 3 hrs, (c) EDC·HCl, HOBt, TEA, 0°C to r.t, 8 hrs. Circular dichroism (CD): The CD spectra of the tripeptide was measured on a JASCO-810 spectropolarimeter under constant nitrogen flow condition. Tripeptide concentration of 200µM in 1 mm path length quartz cuvette was used for all the CD spectra measurements. All the CD measurements were carried out at 25°C with an accuracy of േ0.1. The far-UV region was scanned between 190 to 250 nm using bandwidth of 1 nm. Each represented spectra was the average of three individual scans. UV-vis spectroscopy: The UV-vis absorption spectra of this tripeptide were acquired by using a JASCO V-630 spectrophotometer (JASCO International Co. Ltd, Japan). A high quality quartz cuvette was used for measuring the absorbance of the tripeptide dissolved in methanol between 200 to 500 nm wavelength ranges. FT-IR Spectroscopy: The FT-IR spectra of the tripeptide were recorded on a Bruker TENSOR27 spectrometer by the KBr disc technique. Solid sample was mixed with KBr in a clean glass pestle and compressed to obtain a pellet. Background spectra were obtained with a KBr pellet for each sample. For recording the FT-IR in solution state, tripeptide solution of 200µM concentration in methanol was used. The spectra were scanned from 400–4000 cm-1 at 4 cm-1 resolution. Bruker software was used for data processing.


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Molecular Dynamics: The molecular dynamics (MD) simulations were carried out using Desmond as implemented in Schrödinger Maestro 2015–4 Academic version with 10 ns (nanoseconds) simulation time each. The structure of the tripeptide was drawn in Avogadro and geometry optimized using steepest descent algorithm under UFF force field. Different numbers of molecules (ranging one to twenty) were placed in simulation boxes of the size 90 × 90 × 90 Å3 using packmol. Molecules were placed randomly in the simulation boxes. The final simulation box size in Maestro was 100 × 100 × 100 Å3 to maintain a gap of 10 Å from the periodic boundary. Maestro protein preparation wizard was used to prepare the peptide for simulation. The box was then filled with aqueous medium using simple point charge (SPC) model to describe the water molecules. The whole system was charge neutral. Optimized potentials for liquid simulations (OPLS) 2005 force field was used for the simulations. Five step relaxation protocol was used starting with Brownian dynamics for 100 ps with restraints on solute heavy atoms at NVT (with T = 10 K) followed by 12 ps of dynamics with restraints at NVT (T = 10 K) and then at NPT (T = 10 K) using Berendsen method. Then the temperature was raised to 300 K for 12 ps followed by 24 ps relaxation step without restraints on the solute heavy atoms. The production MD was run at NPT with T = 300 K for 10000 ps (picoseconds). MD results were analysed using the simulation event analyser embedded in Desmond/Maestro. NMR method: 1H and

13

C NMR spectroscopic data were recorded with Bruker DPX 300

MHz and 600 MHz spectrometer. Chemical shifts (δ) were reported in parts per million (ppm) and tetramethylsilane (δ = 0.00) used as the internal standard. All the 1H and 13C NMR spectra of the compound recorded in DMSO-D6 solvent having 1H NMR, δ = 2.50 ppm (s), 13

C NMR, δ = 40 ppm (m) and all data were reported as follows: chemical shift, integration,

multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet) and coupling constant (s) in hertz (Hz). All the experiments were carried out at room temperature (25°C). Atomic force microscope: AFM images of the tripeptide assembly formed after 24 hrs and 48 hrs incubation were obtained on Vecco diCP II with a piezo scanner with the range of 100 µm. The images (256 × 256 pixels) were captured with a scan size between 5 µm and 25 µm at a scan speed rate of 0.5 to 0.6 Hz. Images were processed through flattening via Proscan 1.8 software. AFM image of the tripeptide assembly generated after 6 hrs and 12 hrs incubation were obtained on Pico Plus 5500 AFM (Agilent Technologies, Inc., Santa Clara, CA, USA) with the piezo scanner range of 9 µm. The images (256 × 256 pixels) were captured with a scan size between 0.5 and 5 µm at the scan speed rate of 0.5 rpm. The images



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were processed through flattening via Pico view software (Molecular Imaging Inc., Ann Arbor, MI, USA). RESULTS AND DISCUSSION In this investigation, morphology of the self assembled structure adapted by the tripeptide was determined through atomic force microscopy (AFM), field electron scanning electron microscopy (FESEM). For this purpose, tripeptide dissolved in ethyl acetate and methanol separately drop-casted on the mica film and the specimens were observed under different microscope after drying the sample.

Figure 1. AFM image of the tripeptide (c =0.2 mM) dissolved in ethyl acetate solution and incubated at different time interval (a) Topography of the tripeptide assembly of 25–50 nm size produced after 6 hrs incubation and the inset picture depicted the enlarged view of the tripeptide assembly (b) phase diagram of the mica film corresponding to 25–50 nm sized assembly produced after 6 hrs incubation, (c) topography of the tripeptide assembly of 60– 110 nm size produced after 12 hrs incubation, (d) phase diagram of the mica film corresponding to 60–110 nm sized assembly produced after 12 hrs incubation. From AFM image in figure 1a, it was observed that spherical assembly of 25–50 nm size was produced from the tripeptide (c = 0.2 mM) dissolved in ethyl acetate solvent and incubated for 6 hrs. Figure 1b depicted the phase diagram image of the mica film corresponding to the tripeptide assembly formed after 6 hrs incubation. To check the incubation time dependency of the tripeptide assembly, tripeptide dissolved in ethyl acetate incubated for 12 hrs. Interestingly, it has been found that the size of the spherical assembly increased to 60-110 nm with increasing the aging time from 6 hrs to 12 hrs as shown in figure 1c. The phase diagram image of the mica film corresponding to tripeptide assembly formed after 12 hrs aging are shown in figure 1d. It seems that individual assembly generated after 6 hrs was further aggregated with each other and developed larger sized spherical assembly after 12 hrs aging.


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Aggregation of discrete spherical assembly to larger one with increasing the aging time insisted us to evaluate the effect of incubation time interval of 24 and 48 hrs on the tripeptide assembled structure. Figure 2b portrayed the spherical assembly of 140-200 nm size generated from the ethyl acetate solution of the tripeptide having 0.2 mM concentration aged for 24 hrs. The detailed features of the spherical assembly adapted by the tripeptide after 24 hrs were resolved through the FESEM image. In figure 2a, it has been observed that some of the spherical assembly was formed with a hole inside i.e doughnut shaped assembly with ~300 nm outer diameter and ~100 nm inner diameter, was produced along with spherical assembly from the tripeptide (0.2 mM) dissolved in ethyl acetate solvent and aged for 24 hrs. The assembled structures shown in figure 2a are not uniform in size. It appeared that individual assembly further aggregated with each other and generated doughnut to larger sized spherical assembly. Moreover, it was noticed from fig 2c, that the size of the tripeptide assembly generated after 24 hrs decreased to 60-70 nm after sonication. Therefore sonication possibly breaks the polymer of the assembly and provides the single assembly adopted by the tripeptide in ethyl acetate solution after 24 hrs. This result also corroborates the phenomenon of further aggregation of the discrete assemblies. In addition, pattern of the tripeptide assembly generated after 48 hrs incubation from the tripeptide solution in ethyl acetate has also been evaluated and it was found that the diameter of the spherical assembly increases from 140-200 nm to 600-1000 nm (figure 4c) with increasing the aging time span from 24 hrs to 48 hrs. Therefore, with increasing time span, nano-sphere generated after 24 hrs got the chance for further aggregation and leading to the formation of microsphere. However the pattern of the assembled structure is similar i.e. spherical in four incubation times period (6 hrs, 12 hrs, 24 hrs and 48 hrs). As the tripeptide has strong propensity towards further aggregation of the unit assembly, it may be possible that in concentrated tripeptide solution, aggregation of the assembly led to some other morphology than spherical. For this purpose, concentration dependent self assembly formation was performed. Figure 4c,d showed concentration dependent self assembly pattern of the tripeptide dissolved in ethyl acetate solvent.



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Figure 2. AFM and FESEM image of the tripeptide in ethyl acetate solution after 24 hrs aging. (a) FESEM image of the spherical and doughnut assembly (~ 300 nm) generated from ethyl acetate solution of the tripeptide, (b) AFM image of the spherical assembly generated from the tripeptide dissolved in ethyl acetate (0.2 mM) having size of 140-200 nm, (c) Nanosphere obtained from the ethyl acetate solution of the tripeptide with sonication having size of 60-70 nm. An interesting result has been obtained from the concentration dependent self-assembly study. After 48 hrs incubation, diluted solution of this tripeptide (0.2 mM) produced larger sized i.e. 600-1000 nm spherical assembly (figure 4c), whereas in concentrated solution of the tripeptide (0.6 mM), further fusion of the spherical assembly resulted ring to protofibrillar assembly (figure 4d). Therefore structural transition27 from spherical and annular to protofibril and ring like assembly takes place by changing the concentration of the tripeptide solution. However equilibrium between the spherical and protofibrillar structure is retained throughout the concentration dependent study. Hence the morphology of the assembled structure adapted by this tripeptide is concentration dependent and the size of the spherical assembly is time dependent.

Figure 3. AFM image of the tripeptide (c =0.3 mM) dissolved in methanol solution and incubated at different time interval (a) Topography of the tripeptide assembly of 30–50 nm size produced after 6 hrs incubation, (b) phase diagram of the mica film corresponding to 30–


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50 nm sized assembly produced after 6 hrs incubation, (c) topography of the tripeptide assembly of 70–110 nm size produced after 12 hrs incubation, (d) phase diagram of the mica film corresponding to 70–110 nm sized assembly produced after 12 hrs incubation. Furthermore, self assembly propensity of this tripeptide was investigated in methanol solvent system. For this purpose tripeptide dissolved in methanol incubated for 6 hrs, 12 hrs and 48 hrs respectively. Interestingly it was found that this tripeptide with 0.3 mM concentrated solutions in methanol, produced spherical along with small protofibrillar assembly of 30-50 nm size after 6 hrs aging (figure 3a). Figure 3b depicted the phase diagram image of the mica film corresponding to tripeptide assembly formed after 6 hrs incubation. Subsequently, this tripeptide solution formed spherical assembly of 70-110 nm size after 12 hrs aging as shown in figure 3c. Figure 3d depicted the phase diagram image of the mica film corresponding to tripeptide assembly formed after 12 hrs incubation. Therefore further aggregation of individual assembly for the generation of larger sized assembly with increasing the incubation time period had taken place in methanol solution of this tripeptide. After 48 hrs incubation, this tripeptide solution produced spherical assembly of 600-900 nm size along with some protofibrillar assembly as observed in figure 4a. Generation of protofibrillar assembly from an ordered arrangement of the spherical assembly was clearly observed from the arrow in figure 4a. In addition, concentrated solution of this tripeptide (0.8 mM) generated multilayered spherical shell shaped assembly (figure 4b).

Figure 4. AFM image of the tripeptide dissolved in methanol and ethyl acetate solution separately and aged for 48hrs. (a) assembly generated from diluted (0.3 mM) tripeptide in methanol solution having 600-900 nm size, (b) assembly generated from concentrated (0.8 mM) tripeptide in methanol solution, (c) spherical assembly generated from ethyl acetate solution of the tripeptide (0.2 mM) having size of 600-1000 nm, (d) assembly generated from concentrated tripeptide in ethyl acetate solution (0.6 mM).



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Therefore in both the solvent systems, this tripeptide was able to form self-assembled structure and the pattern of these assembled architectures are similar i.e. spherical and protofibrillar. Hence both the solvent systems offer the similar environment during the selfassembly of this tripeptide i.e. similar type of interactions between this tripeptide and the two solvent systems had taken place during self-assembly and leading to the structurally similar assembly. To assess the size distribution pattern of this tripeptide assembly we plotted the histogram of the assembled tripeptide. From figure 5a it has been clearly observed that, tripeptide generated assembled structure of 600-1000 nm size with maximum intensity at 700-900 nm from the solution of tripeptide dissolved in ethyl acetate with concentration 0.2 mM incubated for 48 hrs whereas tripeptide assembly of 600-900 nm size with maximum intensity at 700-800 nm was developed from the solution of tripeptide dissolved in methanol with concentration 0.3 mM incubated for 48 hrs as observed from figure 5b. Therefore figure 5a and 5b demonstrated the size distribution pattern of the tripeptide assembly in diluted condition after 48 hrs incubation.

Figure 5. Histogram of the size distribution of the tripeptide assembly generated from diluted solution of this tripeptide dissolved in different solvent system. (a) Histogram of the size distribution pattern against concentration of the tripeptide in ethyl acetate solution (C = 0.2 mM), (b) Histogram of the size distribution pattern against concentration of the tripeptide in methanol solution (C = 0.3 mM). In concentrated condition this tripeptide formed protofibril along with globular assembly; for this reason we plotted histogram for diameter as well as length of the tripeptide assembly against concentration of the tripeptide solution. Accordingly, figure 6a depicted that tripeptide dissolved in ethyl acetate with concentration 0.6 mM generated spherical along


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with protofibrillar assembly of 0.5-16 µm length with maximum intensity at 0.5-2 µm after 48 hrs incubation, whereas diameter of these protofibrillar and spherical assembly is within the range of 300-700 nm with maximum intensity at 400-550 nm (figure 6b). Histogram plot for tripeptide dissolved in methanol with concentration 0.8 mM produced layered spherical assembly of 650-1000 nm size with maximum intensity at 700-900 nm after incubating for 48 hrs (figure 6c).

Figure 6. Histogram of the size distribution of the tripeptide assembly generated from concentrated solution of this tripeptide dissolved in different solvent system. (a) Histogram of the length distribution pattern against concentration of the tripeptide in ethyl acetate solution (C = 0.6 mM), (b) Histogram of the diameter distribution pattern against concentration of the tripeptide in ethyl acetate solution (C = 0.6 mM), (c) Histogram of the diameter distribution pattern against concentration of the tripeptide in methanol solution (C = 0.8 mM). Metadynamics were performed on a single tripeptide to elucidate the energetically favoured conformation of the tripeptide backbone in solution state. To fulfil the requirement, 360o scan on each φ-ψ pair were performed and the energy landscape is shown in the figure 7a-d. The area covered by blue color in these heat maps indicates the allowed regions. From this figure, it appears that φ angles in the backbone prefer an orientation of -80o and -150o (figure 7b-d), whereas, the ψ1 take a value of 0o and 150o (figure 7b). ψ2 remains mostly in 60o or 150o orientation (figure 7c) and ψ3 can be in ±60o and ±180o (figure 7d). These indicate that, in the solution state, the peptide backbone may remain in a number of energetically favorable conformations.



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Figure 7. Metadynamics simulation of the peptide backbone. (a) Chemical structure of the tripeptide, the φ-ψ angles is highlighted. (b) Energy landscape for the rotation over φ1 and ψ1. (c) Energy landscape for the rotation over φ2 and ψ2. (d) Energy landscape for the rotation over φ3 and ψ3. In order to elucidate the aggregation process of the tripeptide ensemble in solution state, molecular dynamics28 (MD) stimulations were performed. For this purpose, several randomly organized geometry-optimized tripeptides were simulated in aqueous environment (with SPC water model) for 10 ns using Schrodinger Maestro software. Figure 8 depicted the snapshot of MD stimulation with twenty tripeptide molecules at different time interval. Visualization of assembly formation during the stimulation time period and generation of the most stable assembled structures from twenty disordered tripeptides has been presented through these MD stimulation snapshots (figure 8a-e). Within this limited timescale, five oligomeric assemblies with different population sizes (upto seven) were generated by aggregation of twenty tripeptide molecules. The largest oligomer contained seven tripeptide molecules and the morphology of this aggregate was spherical (figure 8f) which corroborate the result obtained from electron microscopy analysis.


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Figure 8. Snapshots of Molecular Dynamics stimulation on twenty tripeptide molecules in aqueous medium at (a) 0 ns, (b) 2 ns, (c) 5 ns, (d) 7 ns, (e) 10 ns time interval. Standard representation of the colour code for atoms was used: O, red; N, blue; H, white; S, yellow and C, green. (f) Surface representation of the largest assembly composed of seven tripeptides. For surface representation colour code used as: C, grey; H, white; N, blue; S, yellow; O, red. MD stimulation was also performed on a smaller ensemble containing only ten tripeptides. Snapshots of MD stimulation on ten tripeptides (figure 9a) demonstrated aggregation of ten tripeptides into three oligomeric assemblies with the biggest one composed of five tripeptide molecules as shown in figure 9b. Therefore, this MD stimulation analyses established that oligomer size is tripeptide concentration dependent and the possibility of various sized oligomers formation, which are comparable with the results obtained from the AFM and FESEM scanning. Furthermore, existence of the tripeptide in various preferred conformations justified the aggregation propensity of the tripeptide ensemble in solution phase.



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Figure 9. Molecular Dynamics stimulation of ten tripeptide ensemble in aqueous medium after 10 ns, (a) snapshot of ten tripeptide molecules at the time of dissolution, (b) structure of the oligomers developed following self-assembly process after 10 ns. Molecular self assembly process was stabilized by weak interactions, such as van der Waals, hydrophobic, electrostatic, hydrogen bonding and stacking interactions. These reasonably low energy interactions collectively provide intact and well-ordered three dimensional architectures. To get better details about the molecular force promoting the self aggregation process, several experiments were performed.

Figure 10. H/D exchange experiment on tripeptide (10 mM) in DMSO-d6. (a) 1H-NMR spectra of the tripeptide in absence of D2O, (b) 1H-NMR spectra of the tripeptide after 1 mints in presence of 10 µl D2O, (c) 1H-NMR spectra of the tripeptide after 24 hrs in presence of 10 µl D2O. Among them, H/D exchange experiment29 resolved the hydrogen bonding pattern of the three amide protons in solution state. Three amide proton peak are appeared at 7.01 ppm, 8.16-


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8.39 ppm and 8.67 -8.78 ppm respectively for this tripeptide in the NMR spectrum (figure 10a). After addition of 10 µl D2O to a 10 mM tripeptide solution in DMSO-d6, the intensity of the NMR signal of one of the amide proton peak at 7.01 ppm decreases very fast within a minute as shown in figure 10b. However, the other two amide protons remain unperturbed even after 24 hrs (figure 10c), which indicated that these amide protons were engaged in intermolecular H-bond formation with another tripeptide molecule. Therefore between the three amide protons only two protons were engaged with other tripeptide molecules via strong H-bonding, whereas the third amide proton is solvent exposed as it remained noninteracting with the other tripeptide molecules. Different type of interactions associated with the self-assembly process other than hydrogen bonding was investigated by the multidimensional NMR spectroscopy such as ROESY (figure 11). A large number of cross peaks were appeared in ROESY spectra for the tripeptide dissolved in DMSO-D6 solution. These cross peaks were generated due to nonbonded proton-proton interactions present in the tripeptide in solution phase. The observed cross peaks were correlated to the interactions between the (i) proton attached to chiral carbon with the methylene proton (CH2) adjacent to phenyl ring, (ii) aromatic protons with the adjacent methylene proton (CH2), (iii) protons of tertiary butyl group with aromatic proton, (iv) protons of tertiary butyl group with the methylene proton (CH2) adjacent to phenyl ring, (v) amide protons with methylene protons adjacent to sulphur groups, (vi) aromatic protons with methylene protons adjacent to sulphur group, (vii) proton attached to C- terminal chiral carbon with the middle of backbone amide protons, (viii) N-terminal amide proton with C-terminal amide protons. Appearance of several non-bonded proton-proton interactions indicated that the tripeptide interacted with each other and thus stabilized the oligomers formation in the solution phase. Similar type of interactions was observed in the single and self assembled tripeptide structure obtained from the molecular dynamics analysis. Interactions from number (i)-(vii) were intramolecular in nature and interaction (viii) was possibly intermolecular in nature as obtained from the molecular dynamics analysis.



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Figure 11. ROESY spectra of the tripeptide in DMSO-D6 solvent. Several cross peak was observed due to non-bonded proton-proton interactions. Furthermore, the impact of hydrogen bonding and the intermolecular interaction such as π-π stacking on self-aggregation was deduced from molecular dynamics analysis. Although, possibility of existence of one intra-molecular hydrogen bonding in the tripeptide monomer is there, but the number of hydrogen bonding interactions increases with the number of tripeptide molecules involved in oligomerization (figure 12a). This increase in hydrogen bonding interactions with the number of tripeptide molecules participated in the self assembly process indicated that, this self assembly process was motivated by both intra and intermolecular hydrogen bonding between the tripeptide molecules involved in assembly formation. Apart from hydrogen bonding interactions, π-π interactions were also a major driving force for the aggregation of the tripeptide molecules in solution phase. It was observed from the molecular dynamics study that the number of π-π interactions increased with increasing the number of tripeptide participated in self assembly process. From the figure 12b, it has been observed that the single molecule probably unable to undergo intramolecular π-π interactions. However, several number of intermolecular π-π interactions


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were present in self assembled oligomers involving 3, 5 and 7 number of tripeptide molecules respectively.

Figure 12. H-bonding and π-π interaction in tripeptide assembly obtained from the molecular dynamics analysis. (a) Intra and inter molecular hydrogen bond formation in the tripeptide and its oligomers over the 10 ns time scale. (b) Intra and inter molecular π-π stacking interactions in the tripeptide and its oligomers over the 10 ns simulation time. To get better insight into the structural aspects of the tripeptide assembly, we performed quantum mechanical calculation on the oligomer consisting of three tripeptides generated from molecular dynamics stimulation of ten disorganized tripeptides.

Figure 13. Optimized geometry of the tripeptide oligomer (consisting of three tripeptides) using density functional theory. (a) Ball and stick model of the optimized geometry for the assembled structure comprised of three tripeptide molecules. (b) Arrangements of the



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aromatic rings in the same tripeptide assembly highlighted in wireframe representation. Image was rendered in Avogadro. DFT optimized structure of the tripeptide oligomer composed of three tripeptide molecules has been shown as ball and stick model in figure 13a. It has been found that several van der Waals, π-π stacking and hydrogen bonding interactions stabilizes this tripeptide oligomer as observed in the DFT optimized structure represented as wireframe model (figure 13b). Consequently, DFT results corroborated the MD, NMR data regarding the stabilization force for tripeptide assembly. FTIR spectroscopy studies were performed to compare the secondary structures of the tripeptide both in solid and self-assembled state.30 The important FTIR band positions are marked in the figure 14 and assignment of the band positions were provided in supporting information (table S1). In most cases, the characteristic amide I, amide II and amide III bands which are mainly originating from backbone vibrations, appeared at 1600–1690 cm-1, 1480– 1580 cm-1 and 1230– 1300 cm-1 respectively.31 The amide I normal mode originated due to CO stretching vibration and the amide II and amide III correspond to coupling of C-N stretching and N-H in-plane bending. Appearance of the characteristic amide I bands at 1641 cm−1 and 1695 cm−1 and the amide NH stretching band at around 3277 cm−1 (figure 14a) indicated that the tripeptide may exist as intermolecularly hydrogen-bonded extended structure in solid state.32 The

amide II and amide III bands at 1529 and 1242 cm−1

respectively, also corroborate the alignment of the tripeptide as extended conformation in solid state.33 However, significant changes in FTIR band was observed after aging the tripeptide dissolved in methanol solvent. Figure 14b illustrates the FTIR spectra of the tripeptide in the early stage (~20 min after adding to the solvent) of aggregation. The high energy amide I band that appeared at ~1689 cm-1 was relatively broad compared to the band at ~1695 cm-1 in the solid crystalline peptide. It indicated weakening of the –C=O bond, may be due to hydrogen bond formation with the other tripeptide molecules in the assembled state. In addition, the other amide I band shifted from 1641 cm-1 to 1662 cm-1. It broadened further and the band was appeared at 1659 cm-1 after longer (48 hrs) incubation as observed in figure 14c. Shifting of the amide band at 1641 cm-1 to higher frequency ( from 1641 cm-1 to 1659 cm-1) suggested that

this particular -C=O bond associated with this vibration became

stronger in assembled state compared to solid state. However, the ester carbonyl bond frequency was broadened and appeared at 1739 cm-1.


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Figure 14. FTIR spectra of the tripeptide. (a) FTIR Spectra of tripeptide in solid state, (b) FTIR Spectra of tripeptide in methanol solution at 20 minutes, (c) Highlighted the amide I peak in FTIR spectra for the tripeptide dissolved in methanol at 48 hrs. These siftings and broadening of the FTIR bands suggested changes in molecular-molecular interaction pattern as well as the structural rearrangement of the tripeptide aggregated in methanol compared to solid state. Thus the FTIR and NMR data suggested that the tripeptide



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possibly preferred to exist as cross β-sheet unit composed of four intermolecular hydrogen bonds in the aggregated state (figure 15).

Figure 15. Possible hydrogen bonding in the tripeptide in assembled state. UV-vis measurement was recorded on tripeptide dissolved in methanol solvent with various concentrations from 20 µM to 60 µM (figure 16a). UV-vis spectra showed two characteristic peaks at 260 nm and 267 nm for the benzyl groups present in the tripeptide molecule. Scattering was observed which implies self- aggregation of the molecule. Scattering was also observed from the CD spectrum34 for the tripeptide in figure 16b-d.

Figure 16. Absorbance and CD spectra of the tripeptide in solution state, (a) UV-vis spectra of the tripeptide in methanol solution, (b) Circular dichroism spectra of the tripeptide solution (c = 0.2 mM) in methanol at the time of dissolution, (c) Circular dichroism spectra of the


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tripeptide solution in methanol after 2 days, (d) Circular dichroism spectra of the tripeptide solution in methanol after 10 days. Initially the CD measurement for the tripeptide dissolved in methanol showed a negative ellipticity in the far-UV region with a distinct band at 197 nm (figure 16b). Appearance of CD band at 197 nm corresponds to random coil conformation of the tripeptide in solution state at dissolution time. After 48 hrs incubation, the negative ellipticity appeared at 205 nm along with some scattered signal around 211 nm in the CD spectrum (figure 16c). In addition, after 10 days incubation, the negative ellipticity appeared at 206 and the scattered signal 211 remained in the CD spectrum for this tripeptide solution (figure 16d). Therefore, with increasing time, CD band shifted from 197 to 206 nm along with high scattering. This shifting of CD band indicated that peptide conformation changes from random coil to other conformation which was more structured. It seems that, with increasing time span, maximum number of tripeptide molecules tends to form intermolecular hydrogen bonds between the carbonyl oxygen with the amide NH hydrogen and π–π stacking interactions between the aromatic (benzyl) moieties and thus led to other ordered conformation compared to random coil. Conclusion: Oligomeric assembly structures made of different proteins and peptides are implicated in several neurological disorders in recent years. Morphological features are often derived from electron microscopy analysis, however the intra-molecular forces that govern the assembly structure is not well understood. In this context, detail information regarding peptide conformation and interaction patterns is vital to develop some peptide therapeutics. In the current investigation, we presented molecular details of the assembled structure produced by s-benzyl protected cysteine tripeptide. We observed that upon aging the tripeptide in organic solvent it can produce annular and protofibrillar assembly. It was found that weak forces such as hydrogen bonding, π-π stacking, van der Waals and hydrophobic interactions collectively provide intact and well-ordered three dimensional architectures. Based on the NMR, FTIR and molecular dynamic analysis we showed that the tripeptide may prefer a cross beta sheet pattern in the assembled structure. These molecular details may aid to design peptide based therapeutics for amyloid disorder including Alzheimer’s and Parkinson disease. Supporting Information: The Supporting Information section includes characterization data, mass spectra, COSY, HSQC NMR spectral data, FTIR spectra along with band



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assignment table and the coordinates of DFT optimization for the tripeptide. This information is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author Correspondence should be addressed to *Biswadip Banerji: phone, (+) 91 33 24995709; fax, (+) 91 33 24735197; email, [email protected], address: Department of Organic & Medicinal Chemistry, CSIR-Indian Institute of Chemical Biology; 4, Raja S.C. Mullick Road, Kolkata, and Country. India-700032 *Nakul Chandra Maiti: phone, +91-33-2499-5940; fax, +91-33-2473-5197; email, [email protected], address: Structural Biology and BioInformatics, CSIR-Indian Institute of Chemical Biology; 4, Raja S.C. Mullick Road, Kolkata, and Country. India-700032

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. Acknowledgments: M.C. thanks University Grants Commission, India, for financial support. U.P. thanks INSPIRE fellowship program, Department of Science and Technology, Government of India, for financial support. The authors would also like to thank the central instrumentation facilities of CSIR-Indian Institute of Chemical Biology for recording the spectra. We also acknowledge project GAP-299, DBT, New Delhi and the CSIR network projects BSC0113, BSC0115 and BSC0121 for funding and related support. References (1)

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