Research Article Received: 15 September 2013
Revised: 19 November 2013
Accepted: 26 November 2013
Published online in Wiley Online Library: 30 January 2014
(wileyonlinelibrary.com) DOI 10.1002/psc.2606
Nanostructures from the self-assembly of α-helical peptide amphiphiles Qingbin Meng,† Yingying Kou,† Xin Ma, Lei Guo and Keliang Liu* Self-assembly of PAs composed of palmitic acid and several repeated heptad peptide sequences, C15H31CO-(IEEYTKK)n-NH2 (n = 1–4, represented by PA1–PA4), was investigated systematically. The secondary structures of the PAs were characterized by CD. PA3 and PA4 (n = 3 and 4, respectively) showed an α-helical structure, whereas PA1 and PA2 (n = 1 and 2, respectively) did not display an α-helical conformations under the tested conditions. The morphology of the self-assembled peptides in aqueous medium was studied by transmission electron microscopy. As the number of heptad repeats in the PAs increased, the nanostructure of the self-assembled peptides changed from nanofibers to nanovesicles. Changes of the secondary structures and the self-assembly morphologies of PA3 and PA4 in aqueous medium with various cations were also studied. The critical micelle concentrations were determined using a pyrene fluorescence probe. In conclusion, this method may be used to design new peptide nanomaterials. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. Keywords: peptide amphiphiles; self-assembly; nanostructures; α-helical
Introduction
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* Correspondence to: Keliang Liu, Beijing Institute of Pharmacology and Toxicology, Beijing, 100850, China. E-mail: [email protected] †
These authors contributed equally to this work. Beijing Institute of Pharmacology and Toxicology, Beijing, 100850, China Abbreviations: PAs, peptide amphiphiles; HBTU, O-(1H-benzotriazole-1-yl)-N, N,N′,N′-tetramethyluronium hexafluorophosphate; TEM, transmission electron microscopy; CMC, critical micelle concentration.
Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
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It is well known that α-helical structures play an important role in determining protein structures and functions [1]. Peptides with α-helical secondary structure are important building blocks in the field of molecular self-assembly [2,3]. Some nanostructures are made by using peptides with α-helical conformation. For example, Baumann et al. [4] have reported the self-assembly of cationichead surfactant-like eight-residue peptides. The supramolecules for random-coil and α-helical peptides self-assembled into rod structures, while β-sheet peptides correlated with ribbons. The peptide L6K2 showed the most shallow adsorbed structure height because the great helix propensity led to a shortened monomer length. In addition, Gribbon et al. [5] have reported that a peptide based on an α-helical coiled-coil motif self-assembled into extended and thickened nano-to-mesoscale fibers of high stability and order. These results provided a good framework for the development of future functional biomaterials. The α-helical conformation can be obtained by elaborate peptide sequence design [6]. Furthermore, covalent conjugation of monoalkyl hydrocarbon chains to peptides also can induce and stabilize α-helical secondary and tertiary structures of peptides [7,8]. The design of self-assembled peptides with α-helical conformation based on the relatively well-understood leucine-zipper motif has been reported previously [9–11]. However, the self-assembly of alkyl-modified peptides containing α-helical secondary structure has been rarely studied. The conjugation of peptides with hydrophobic nonpeptide tails [12], called PAs, has been intensively studied for their self-assembly abilities due to their potential structural diversity [13,14] as well as their broad applications in the fields of biotechnology [15–17], tissue engineering [18–20], drug delivery [21,22], regenerative medicine [23], and bacterial inhibition [24]. The amphiphilic nature of the molecules enables the presence of hydrophilic peptides on the surface of the assembled nanostructures, while the hydrophobic tails are confined to the core. PAs can self-assemble into a number of different structures including micelles, vesicles, and nanofibers, depending
on concentration, ionic strength, pH value, temperature, and so on [25,26]. Stupp and coworkers [27–32] have synthesized a broad range of PAs containing β-sheet secondary structures to produce self-assembling biomaterials in aqueous medium with great potential for tissue engineering and drug delivery applications. In addition, Shimada et al. [33] have reported that the self-assembled micelles of the PA C16-W3K (C15H31CO-WA4KA4KA4KA-NH2) altered their morphology from spheres to nanofibers with a simultaneous secondary structure change from a mixture of α-helical and random-coil structures to mainly β-sheets. Furthermore, Malkar et al. [34] investigated the changes in α-helicity that occurred when peptides were modified with alkyl chains ranging from 6 to 18 carbon atoms long. The self-assembly of α-helical PAs is a worthy challenge, and the creation of functional nanostructures has attracted considerable attention. Previously, we designed and synthesized a series of α-helical peptides with heptad repeats to study their HIV-1 cell fusion inhibitory activity [35]. Most of the peptides showed high α-helicity. The heptad repeat (denoted a-b-c-d-e-f-g) was a typical unit in α-helical coiled-coil. Positions a and d are often occupied by hydrophobic residues, while positions e and g are often occupied by charged residues or residues with polar side chain. Such distribution of amino acid residue made the helices amphiphilic with a and d residues forming inter-helical hydrophobic core and residues at e and g form inter-helical ionic interactions [36]. In this paper, one of the heptad repeat sequences,
MENG ET AL. IEEYTKK, was selected as the lead peptide sequence for the design of PAs to investigate the self-assembly of α-helical peptides. In order to induce α-helix formation and form self-assembled structures, a palmitic acid group was coupled to the N-terminus of the peptides containing one to four heptad repeat sequences [35] to form C15H31CO-(IEEYTKK)n-NH2 (n = 1–4), named PA1–PA4. We systematically investigated the self-assembly behavior of the PAs by varying the peptide lengths and assaying them under different conditions.
Materials and Methods Protected amino acids (Fmoc-Ile-OH, Fmoc-Glu(tBu)-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Thr(tBu)-OH, and Fmoc-Lys(Boc)-OH), HBTU, and anhydrous N-hydroxybenzotriazole were purchased from GL Biochem (Shanghai, China) Ltd. and used as received. Rink amide resin (from Tianjing Nankai Hecheng Sci. & Tech. Co. Ltd., Tianjin, China) and N, N′-diisopropylethylamine (DIEA, Acros, Geel, Belgium) were used as received. Pyrene was obtained from Alfa Aesar (Heysham, UK) and recrystallized twice. Palmitic acid and other reagents were of analytical grade or better and were used without further purification. Water was obtained from a Millipore (Molsheim, France) water purification system and had a minimum resistivity of 18.2 MΩ·cm. Synthesis and Purification of Peptides Peptides were synthesized on rink amide resin via a CEM Liberty microwave automated synthesizer (Matthews, NC, USA) employing the standard Fmoc-protocol and HBTU activation. The peptide resin was divided into two portions: one of which was acetylated and the other was modified with palmitic acid at the N-terminus. The resulting resin-bound peptides were cleaved; at the same time, side-chain residues were deprotected for 2 h using a mixed solution of TFA, m-cresol, and water (90 : 5 : 5). Excess TFA was removed by rotary evaporation, and the resulting viscous peptide solution was triturated with cold diethyl ether. The white precipitate was collected and dried in vacuo, redissolved in water, and purified using RP-HPLC to >90% homogeneity on a C18 peptide/protein column. The molecular weight of the peptide was confirmed using MALDI-TOF mass spectrophotometry (Autoflex III, Bruker Daltonics, Bremen, Germany). Circular Dichroism The CD experiments were performed on a BioLogic MOS-450 spectropolarimeter (Claix, France) at room temperature. Solutions of each peptide at 0.1 mg/ml concentration were freshly prepared in pure water. A 400-μl sample of the peptide solution was placed in a 1-mm path length quartz cell. The spectra averaged over three scans were obtained between 280 and 190 nm at a resolution of 0.5 nm and a scan rate of 300 nm/min.
Critical Micelle Concentration Pyrene was used as the fluorescence probe to investigate the CMC. PA solutions were prepared at various concentrations between 1μM and 3 mM and containing 2 μM pyrene. The fluorescence spectra of the solutions were recorded using an LS-55 luminescence spectrometer (Perkin Elmer Co., Waltham, MA, USA) at an excitation wavelength of 334 nm, an excitation slit width of 4 nm, and an emission slit width of 3 nm. The region from 340 to 450 nm was examined at a scan speed of 300 nm/min. A total of five characteristic peaks were observed for each sample. The peak near 373 nm (I1) and the peak near 383 nm (I3) of all solutions were recorded.
Results and Discussion Corey–Pauling–Koltun models of the PAs [C15H31CO-(IEEYTKK)n-NH2 (n = 1–4)] are depicted in Figure 1. Each PA had a hydrophobic alkyl chain tail of palmitic acid and a hydrophilic peptide head group consisting of 1–4 heptad repeats. To determine the effect of alkyl chain conjugation on the secondary structure, CD spectroscopy was used to investigate the conformations of Ac-(IEEYTKK)n-NH2 (n = 1–4) and PAs 1–4. The peptides Ac-(IEEYTKK)n-NH2 (n = 1 and 2) had a random-coil geometry (Figure 2(A)) as shown by the negative absorption peak near 200 nm in the CD spectra, which has been previously attributed to the random-coil structure [37]. As shown in Figure 2(B), the CD spectra suggested that PA3 and PA4 obviously contained an α-helical structure, while PA1 and PA2 adopted β-sheet and random-coil structures due to the negative absorption peak at about 218 nm. The α-helical contents were calculated according to the following equations and are shown in Table 1 [38]. f H ¼ ðθ222 –θC Þ=ðθH –θC Þ θH ¼ ð–4400 þ 250T Þð1–3=Nr Þ θC ¼ 2220–53T where T is the experimental temperature (°C) and Nr is the number of residues in the peptide chain. θC and θH are the baseline ellipticities of random coil and total helix, respectively. The α-helical contents of PA3 and PA4 were greater than those of the peptides Ac-(IEEYTKK)n-NH2 (n = 3 and 4). These results indicated that increasing the number of heptad repeats and conjugation of the hydrophobic alkyl chain both improved the α-helical content, which agreed well with previous reports [39]. We hypothesized that the aggregation of PAs induced by
Transmission Electron Microscopy
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The morphology of the self-assembled PAs was observed using an H-7650 TEM at an accelerating voltage of 80 kV. The PA solution samples were prepared by deposition for 10 min on a copper TEM grid with a holey carbon support film, stained for 2 min by 2% (w/v) phosphotungstic acid solution, and then held in place with tweezers mounted to a Vitrobot (HITACHI, Tokyo, Japan). Images were acquired using a AMT16000 charge-coupled device (CCD) camera (Woburn, MA, USA).
Figure 1. Corey–Pauling–Koltun models of PAs 1–4 in extended conformation.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2014; 20: 223–228
NANOSTRUCTURES FROM THE SELF-ASSEMBLY OF α-HELICAL PEPTIDE AMPHIPHILES
Figure 2. CD spectra of (A) Ac-(IEEYTKK)n-NH2 (n = 1–4) and (B) PAs 1–4 in 0.1 mg/ml aqueous mediums.
Table 1. α-helical contents of PAs and Ac-(IEEYTKK)n-NH2 (n = 3 and 4) Sample name Ac-(IEEYTKK)3-NH2 PA3 Ac-(IEEYTKK)4-NH2 PA4
Peptide sequence Ac-(IEEYTKK)3-NH2 C15H31CO-(IEEYTKK)3-NH2 Ac-(IEEYTKK)4-NH2 C15H31CO-(IEEYTKK)4-NH2
Content of α-helices (%) 28.05 36.21 35.13 42.22
hydrophobic alkyl chains brought the peptide segments into closer proximity and increased the effective concentration [7]. As shown in Figure 3, PA1 and PA2 self-assembled into tightly packed nanofibers in aqueous medium, while PA3 and PA4 formed
spherical micelles and nanovesicles. Thus, the morphology of self-assembled PAs changed from nanofibers to spherical micelles when the length of the peptides increased. The hydrophilicity of the PAs increased as the peptide length increased, resulting in relatively loose self-assembled nanostructures due to the combined effects of steric hindrance and electrostatic interactions. The CMC is a key parameter that describes the physical properties and thermodynamic stability of micelles [40]. The CMCs of PAs in aqueous medium were measured using pyrene probe fluorescence spectrometry, which is a recognized method of determining CMC values [41,42]. As presented in Figure 4(A), I1 and I3 are the emission intensities of the first and third bands in the fluorescence spectrum of pyrene under high resolution. The emission intensity ratio of I3/I1 is very sensitive to the polarity of the medium surrounding the pyrene molecules, with a smaller
Figure 3. TEM images of (A) PA1, (B) PA2, (C) PA3, and (D) PA4 in 0.1 mg/ml aqueous mediums.
J. Pept. Sci. 2014; 20: 223–228 Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci
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Figure 4. (A) Fluorescence spectra of pyrene (2 μM) in various concentrations of PA1. (B) Relationship of intensity ratio (I3/I1) and concentrations of PA solutions ratio (I3/I1) and concentrations of PA solutions.
MENG ET AL. ratio indicating greater polarity. The relationship between I3/I1 and PA concentration is depicted in Figure 4(B). All of the curves exhibited sigmoidal shapes [43]. The intensity ratio of I3/I1 remained nearly unchanged at low PA concentrations, indicating that pyrene was located in an aqueous environment. As the PA concentration increased, I3/I1 dramatically increased to the level characteristic of pyrene in a completely hydrophobic
Table 2. CMC values of PAs Sample name PA1 PA2 PA3 PA4
Peptide sequence C15H31CO-(IEEYTKK)1-NH2 C15H31CO-(IEEYTKK)2-NH2 C15H31CO-(IEEYTKK)3-NH2 C15H31CO-(IEEYTKK)4-NH2
CMC(μM) 5.62 6.76 10.14 11.17
environment, suggesting the formation of micelles or other structures with a hydrophobic interior. The CMCs were obtained from the sigmoidal curve and are summarized in Table 2. The CMC values increased as the number of heptad repeats of the peptide sequence increased, owing to the hydrophilic nature of the peptide segment. In order to study the self-assembly mechanism of PAs with α-helical secondary structure, we investigated the morphology of the PA3 and PA4 structures formed in solutions with various cations [44]. Figure 5(I) shows the conversion of nanostructures from spherical micelles to cylindrical nanofibers coexisting with spherical micelles when 10 mM Ca2+ was added to the PA3 solution. The self-assembled morphology of PA4 still retained the nanovesicles, but the size decreased when 10 mM Na+ was added. However, the morphology of PA4 changed from vesicles to spherical micelles upon the addition of 10 mM Ca2+ (Figure 5(II)). Thus, the addition of cations destroyed the electrostatic equilibrium of PA3 and PA4
Figure 5. TEM images of 0.1 mg/ml PA solutions containing (A) no additional cations, (B) 10 mM NaCl, and (C) 10 mM CaCl2 for (I) PA3 and (II) PA4.
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Figure 6. CD spectra of 0.1 mg/ml PA solutions with 10 mM different cations: (A) PA3 and (B) PA4.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2014; 20: 223–228
NANOSTRUCTURES FROM THE SELF-ASSEMBLY OF α-HELICAL PEPTIDE AMPHIPHILES
Figure 7. Schematic illustration of the self-assembly of PAs 1–4 in aqueous mediums.
the hydrophilic segments. According to a previous report [44], the presence of hydrophobic interactions from the alkyl tails has an ability to protect the self-assembled nanostructures. Thus, in this study, PA3 formed nanofibers in the presence of CaCl2 but formed spherical micelles under other conditions.
Conclusion We designed a series of four PAs with varied peptide head group lengths. PA1 and PA2 formed tightly packed nanofibers, while PA3 and PA4 formed loose spherical micelles and nanovesicles. The CMC values of PAs increased with longer peptides, indicating that the nanostructure morphology and size were influenced by the hydrophilic-to-hydrophobic ratio [44,49]. Cylindrical nanofibers coexisting with spherical micelles were formed in PA3 solutions in the presence of 10 mM Ca2+. Hydrophobic interactions between the alkyl tails were the main driving force for the formation of self-assembled nanostructures, while electrostatic interactions and steric hindrance also played important roles in the overall self-assembly. This work suggests that the self-assembly of α-helical PAs may be controlled by the hydrophilic-to-hydrophobic ratio and ionic strength. This valuable research provides a useful way of modulating the self-assembly of α-helical PAs.
J. Pept. Sci. 2014; 20: 223–228 Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci
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and caused a tighter array of the peptide segment [45]. The screening of electrostatic repulsion by the metal cations generated an enormous effect on the self-assembly of PAs, resulting in a transition of the morphology of the self-assembled structures. The divalent cation Ca2+ played a greater role to induce the PAs to form tight aggregations compared with the monovalent cation Na+. The CD spectra of PA3 and PA4 in the presence of Na+ and Ca2+ are shown in Figure 6. The α-helical secondary structures of PA3 and PA4 were gradually transformed to β-sheets upon cation (Na+ or Ca2+) addition, revealing a change from the original conformation. A proposed PA self-assembly mechanism is illustrated in Figure 7. Based on previous reports [44,46–48], the driving forces governing the self-assembly of PAs in aqueous medium arise from several combined effects, including hydrophobic interactions of the alkyl tails, steric hindrance effects, electrostatic interactions, and hydrogen bonding among the peptide sequences. The final structural properties such as size and shape reflect a delicate balance of these effects. In PA1 and PA2, hydrophobic interactions between the alkyl tails were the main driving force for the formation of nanofiber structures, and the relatively attenuated hydrophobic interactions of PA3 and PA4 led to the formation of spherical micelles [44]. Increasing the length of the peptides could gradually strengthen the interactions with
MENG ET AL. Acknowledgements This work was supported by the National Natural Science Foundation of China (81202465 and 81373266) and the National Key Technologies R & D Program for New Drugs of China (2012ZX09301003).
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wileyonlinelibrary.com/journal/jpepsci Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2014; 20: 223–228
Revised: 19 November 2013
Accepted: 26 November 2013
Published online in Wiley Online Library: 30 January 2014
(wileyonlinelibrary.com) DOI 10.1002/psc.2606
Nanostructures from the self-assembly of α-helical peptide amphiphiles Qingbin Meng,† Yingying Kou,† Xin Ma, Lei Guo and Keliang Liu* Self-assembly of PAs composed of palmitic acid and several repeated heptad peptide sequences, C15H31CO-(IEEYTKK)n-NH2 (n = 1–4, represented by PA1–PA4), was investigated systematically. The secondary structures of the PAs were characterized by CD. PA3 and PA4 (n = 3 and 4, respectively) showed an α-helical structure, whereas PA1 and PA2 (n = 1 and 2, respectively) did not display an α-helical conformations under the tested conditions. The morphology of the self-assembled peptides in aqueous medium was studied by transmission electron microscopy. As the number of heptad repeats in the PAs increased, the nanostructure of the self-assembled peptides changed from nanofibers to nanovesicles. Changes of the secondary structures and the self-assembly morphologies of PA3 and PA4 in aqueous medium with various cations were also studied. The critical micelle concentrations were determined using a pyrene fluorescence probe. In conclusion, this method may be used to design new peptide nanomaterials. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. Keywords: peptide amphiphiles; self-assembly; nanostructures; α-helical
Introduction
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* Correspondence to: Keliang Liu, Beijing Institute of Pharmacology and Toxicology, Beijing, 100850, China. E-mail: [email protected] †
These authors contributed equally to this work. Beijing Institute of Pharmacology and Toxicology, Beijing, 100850, China Abbreviations: PAs, peptide amphiphiles; HBTU, O-(1H-benzotriazole-1-yl)-N, N,N′,N′-tetramethyluronium hexafluorophosphate; TEM, transmission electron microscopy; CMC, critical micelle concentration.
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It is well known that α-helical structures play an important role in determining protein structures and functions [1]. Peptides with α-helical secondary structure are important building blocks in the field of molecular self-assembly [2,3]. Some nanostructures are made by using peptides with α-helical conformation. For example, Baumann et al. [4] have reported the self-assembly of cationichead surfactant-like eight-residue peptides. The supramolecules for random-coil and α-helical peptides self-assembled into rod structures, while β-sheet peptides correlated with ribbons. The peptide L6K2 showed the most shallow adsorbed structure height because the great helix propensity led to a shortened monomer length. In addition, Gribbon et al. [5] have reported that a peptide based on an α-helical coiled-coil motif self-assembled into extended and thickened nano-to-mesoscale fibers of high stability and order. These results provided a good framework for the development of future functional biomaterials. The α-helical conformation can be obtained by elaborate peptide sequence design [6]. Furthermore, covalent conjugation of monoalkyl hydrocarbon chains to peptides also can induce and stabilize α-helical secondary and tertiary structures of peptides [7,8]. The design of self-assembled peptides with α-helical conformation based on the relatively well-understood leucine-zipper motif has been reported previously [9–11]. However, the self-assembly of alkyl-modified peptides containing α-helical secondary structure has been rarely studied. The conjugation of peptides with hydrophobic nonpeptide tails [12], called PAs, has been intensively studied for their self-assembly abilities due to their potential structural diversity [13,14] as well as their broad applications in the fields of biotechnology [15–17], tissue engineering [18–20], drug delivery [21,22], regenerative medicine [23], and bacterial inhibition [24]. The amphiphilic nature of the molecules enables the presence of hydrophilic peptides on the surface of the assembled nanostructures, while the hydrophobic tails are confined to the core. PAs can self-assemble into a number of different structures including micelles, vesicles, and nanofibers, depending
on concentration, ionic strength, pH value, temperature, and so on [25,26]. Stupp and coworkers [27–32] have synthesized a broad range of PAs containing β-sheet secondary structures to produce self-assembling biomaterials in aqueous medium with great potential for tissue engineering and drug delivery applications. In addition, Shimada et al. [33] have reported that the self-assembled micelles of the PA C16-W3K (C15H31CO-WA4KA4KA4KA-NH2) altered their morphology from spheres to nanofibers with a simultaneous secondary structure change from a mixture of α-helical and random-coil structures to mainly β-sheets. Furthermore, Malkar et al. [34] investigated the changes in α-helicity that occurred when peptides were modified with alkyl chains ranging from 6 to 18 carbon atoms long. The self-assembly of α-helical PAs is a worthy challenge, and the creation of functional nanostructures has attracted considerable attention. Previously, we designed and synthesized a series of α-helical peptides with heptad repeats to study their HIV-1 cell fusion inhibitory activity [35]. Most of the peptides showed high α-helicity. The heptad repeat (denoted a-b-c-d-e-f-g) was a typical unit in α-helical coiled-coil. Positions a and d are often occupied by hydrophobic residues, while positions e and g are often occupied by charged residues or residues with polar side chain. Such distribution of amino acid residue made the helices amphiphilic with a and d residues forming inter-helical hydrophobic core and residues at e and g form inter-helical ionic interactions [36]. In this paper, one of the heptad repeat sequences,
MENG ET AL. IEEYTKK, was selected as the lead peptide sequence for the design of PAs to investigate the self-assembly of α-helical peptides. In order to induce α-helix formation and form self-assembled structures, a palmitic acid group was coupled to the N-terminus of the peptides containing one to four heptad repeat sequences [35] to form C15H31CO-(IEEYTKK)n-NH2 (n = 1–4), named PA1–PA4. We systematically investigated the self-assembly behavior of the PAs by varying the peptide lengths and assaying them under different conditions.
Materials and Methods Protected amino acids (Fmoc-Ile-OH, Fmoc-Glu(tBu)-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Thr(tBu)-OH, and Fmoc-Lys(Boc)-OH), HBTU, and anhydrous N-hydroxybenzotriazole were purchased from GL Biochem (Shanghai, China) Ltd. and used as received. Rink amide resin (from Tianjing Nankai Hecheng Sci. & Tech. Co. Ltd., Tianjin, China) and N, N′-diisopropylethylamine (DIEA, Acros, Geel, Belgium) were used as received. Pyrene was obtained from Alfa Aesar (Heysham, UK) and recrystallized twice. Palmitic acid and other reagents were of analytical grade or better and were used without further purification. Water was obtained from a Millipore (Molsheim, France) water purification system and had a minimum resistivity of 18.2 MΩ·cm. Synthesis and Purification of Peptides Peptides were synthesized on rink amide resin via a CEM Liberty microwave automated synthesizer (Matthews, NC, USA) employing the standard Fmoc-protocol and HBTU activation. The peptide resin was divided into two portions: one of which was acetylated and the other was modified with palmitic acid at the N-terminus. The resulting resin-bound peptides were cleaved; at the same time, side-chain residues were deprotected for 2 h using a mixed solution of TFA, m-cresol, and water (90 : 5 : 5). Excess TFA was removed by rotary evaporation, and the resulting viscous peptide solution was triturated with cold diethyl ether. The white precipitate was collected and dried in vacuo, redissolved in water, and purified using RP-HPLC to >90% homogeneity on a C18 peptide/protein column. The molecular weight of the peptide was confirmed using MALDI-TOF mass spectrophotometry (Autoflex III, Bruker Daltonics, Bremen, Germany). Circular Dichroism The CD experiments were performed on a BioLogic MOS-450 spectropolarimeter (Claix, France) at room temperature. Solutions of each peptide at 0.1 mg/ml concentration were freshly prepared in pure water. A 400-μl sample of the peptide solution was placed in a 1-mm path length quartz cell. The spectra averaged over three scans were obtained between 280 and 190 nm at a resolution of 0.5 nm and a scan rate of 300 nm/min.
Critical Micelle Concentration Pyrene was used as the fluorescence probe to investigate the CMC. PA solutions were prepared at various concentrations between 1μM and 3 mM and containing 2 μM pyrene. The fluorescence spectra of the solutions were recorded using an LS-55 luminescence spectrometer (Perkin Elmer Co., Waltham, MA, USA) at an excitation wavelength of 334 nm, an excitation slit width of 4 nm, and an emission slit width of 3 nm. The region from 340 to 450 nm was examined at a scan speed of 300 nm/min. A total of five characteristic peaks were observed for each sample. The peak near 373 nm (I1) and the peak near 383 nm (I3) of all solutions were recorded.
Results and Discussion Corey–Pauling–Koltun models of the PAs [C15H31CO-(IEEYTKK)n-NH2 (n = 1–4)] are depicted in Figure 1. Each PA had a hydrophobic alkyl chain tail of palmitic acid and a hydrophilic peptide head group consisting of 1–4 heptad repeats. To determine the effect of alkyl chain conjugation on the secondary structure, CD spectroscopy was used to investigate the conformations of Ac-(IEEYTKK)n-NH2 (n = 1–4) and PAs 1–4. The peptides Ac-(IEEYTKK)n-NH2 (n = 1 and 2) had a random-coil geometry (Figure 2(A)) as shown by the negative absorption peak near 200 nm in the CD spectra, which has been previously attributed to the random-coil structure [37]. As shown in Figure 2(B), the CD spectra suggested that PA3 and PA4 obviously contained an α-helical structure, while PA1 and PA2 adopted β-sheet and random-coil structures due to the negative absorption peak at about 218 nm. The α-helical contents were calculated according to the following equations and are shown in Table 1 [38]. f H ¼ ðθ222 –θC Þ=ðθH –θC Þ θH ¼ ð–4400 þ 250T Þð1–3=Nr Þ θC ¼ 2220–53T where T is the experimental temperature (°C) and Nr is the number of residues in the peptide chain. θC and θH are the baseline ellipticities of random coil and total helix, respectively. The α-helical contents of PA3 and PA4 were greater than those of the peptides Ac-(IEEYTKK)n-NH2 (n = 3 and 4). These results indicated that increasing the number of heptad repeats and conjugation of the hydrophobic alkyl chain both improved the α-helical content, which agreed well with previous reports [39]. We hypothesized that the aggregation of PAs induced by
Transmission Electron Microscopy
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The morphology of the self-assembled PAs was observed using an H-7650 TEM at an accelerating voltage of 80 kV. The PA solution samples were prepared by deposition for 10 min on a copper TEM grid with a holey carbon support film, stained for 2 min by 2% (w/v) phosphotungstic acid solution, and then held in place with tweezers mounted to a Vitrobot (HITACHI, Tokyo, Japan). Images were acquired using a AMT16000 charge-coupled device (CCD) camera (Woburn, MA, USA).
Figure 1. Corey–Pauling–Koltun models of PAs 1–4 in extended conformation.
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NANOSTRUCTURES FROM THE SELF-ASSEMBLY OF α-HELICAL PEPTIDE AMPHIPHILES
Figure 2. CD spectra of (A) Ac-(IEEYTKK)n-NH2 (n = 1–4) and (B) PAs 1–4 in 0.1 mg/ml aqueous mediums.
Table 1. α-helical contents of PAs and Ac-(IEEYTKK)n-NH2 (n = 3 and 4) Sample name Ac-(IEEYTKK)3-NH2 PA3 Ac-(IEEYTKK)4-NH2 PA4
Peptide sequence Ac-(IEEYTKK)3-NH2 C15H31CO-(IEEYTKK)3-NH2 Ac-(IEEYTKK)4-NH2 C15H31CO-(IEEYTKK)4-NH2
Content of α-helices (%) 28.05 36.21 35.13 42.22
hydrophobic alkyl chains brought the peptide segments into closer proximity and increased the effective concentration [7]. As shown in Figure 3, PA1 and PA2 self-assembled into tightly packed nanofibers in aqueous medium, while PA3 and PA4 formed
spherical micelles and nanovesicles. Thus, the morphology of self-assembled PAs changed from nanofibers to spherical micelles when the length of the peptides increased. The hydrophilicity of the PAs increased as the peptide length increased, resulting in relatively loose self-assembled nanostructures due to the combined effects of steric hindrance and electrostatic interactions. The CMC is a key parameter that describes the physical properties and thermodynamic stability of micelles [40]. The CMCs of PAs in aqueous medium were measured using pyrene probe fluorescence spectrometry, which is a recognized method of determining CMC values [41,42]. As presented in Figure 4(A), I1 and I3 are the emission intensities of the first and third bands in the fluorescence spectrum of pyrene under high resolution. The emission intensity ratio of I3/I1 is very sensitive to the polarity of the medium surrounding the pyrene molecules, with a smaller
Figure 3. TEM images of (A) PA1, (B) PA2, (C) PA3, and (D) PA4 in 0.1 mg/ml aqueous mediums.
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Figure 4. (A) Fluorescence spectra of pyrene (2 μM) in various concentrations of PA1. (B) Relationship of intensity ratio (I3/I1) and concentrations of PA solutions ratio (I3/I1) and concentrations of PA solutions.
MENG ET AL. ratio indicating greater polarity. The relationship between I3/I1 and PA concentration is depicted in Figure 4(B). All of the curves exhibited sigmoidal shapes [43]. The intensity ratio of I3/I1 remained nearly unchanged at low PA concentrations, indicating that pyrene was located in an aqueous environment. As the PA concentration increased, I3/I1 dramatically increased to the level characteristic of pyrene in a completely hydrophobic
Table 2. CMC values of PAs Sample name PA1 PA2 PA3 PA4
Peptide sequence C15H31CO-(IEEYTKK)1-NH2 C15H31CO-(IEEYTKK)2-NH2 C15H31CO-(IEEYTKK)3-NH2 C15H31CO-(IEEYTKK)4-NH2
CMC(μM) 5.62 6.76 10.14 11.17
environment, suggesting the formation of micelles or other structures with a hydrophobic interior. The CMCs were obtained from the sigmoidal curve and are summarized in Table 2. The CMC values increased as the number of heptad repeats of the peptide sequence increased, owing to the hydrophilic nature of the peptide segment. In order to study the self-assembly mechanism of PAs with α-helical secondary structure, we investigated the morphology of the PA3 and PA4 structures formed in solutions with various cations [44]. Figure 5(I) shows the conversion of nanostructures from spherical micelles to cylindrical nanofibers coexisting with spherical micelles when 10 mM Ca2+ was added to the PA3 solution. The self-assembled morphology of PA4 still retained the nanovesicles, but the size decreased when 10 mM Na+ was added. However, the morphology of PA4 changed from vesicles to spherical micelles upon the addition of 10 mM Ca2+ (Figure 5(II)). Thus, the addition of cations destroyed the electrostatic equilibrium of PA3 and PA4
Figure 5. TEM images of 0.1 mg/ml PA solutions containing (A) no additional cations, (B) 10 mM NaCl, and (C) 10 mM CaCl2 for (I) PA3 and (II) PA4.
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Figure 6. CD spectra of 0.1 mg/ml PA solutions with 10 mM different cations: (A) PA3 and (B) PA4.
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NANOSTRUCTURES FROM THE SELF-ASSEMBLY OF α-HELICAL PEPTIDE AMPHIPHILES
Figure 7. Schematic illustration of the self-assembly of PAs 1–4 in aqueous mediums.
the hydrophilic segments. According to a previous report [44], the presence of hydrophobic interactions from the alkyl tails has an ability to protect the self-assembled nanostructures. Thus, in this study, PA3 formed nanofibers in the presence of CaCl2 but formed spherical micelles under other conditions.
Conclusion We designed a series of four PAs with varied peptide head group lengths. PA1 and PA2 formed tightly packed nanofibers, while PA3 and PA4 formed loose spherical micelles and nanovesicles. The CMC values of PAs increased with longer peptides, indicating that the nanostructure morphology and size were influenced by the hydrophilic-to-hydrophobic ratio [44,49]. Cylindrical nanofibers coexisting with spherical micelles were formed in PA3 solutions in the presence of 10 mM Ca2+. Hydrophobic interactions between the alkyl tails were the main driving force for the formation of self-assembled nanostructures, while electrostatic interactions and steric hindrance also played important roles in the overall self-assembly. This work suggests that the self-assembly of α-helical PAs may be controlled by the hydrophilic-to-hydrophobic ratio and ionic strength. This valuable research provides a useful way of modulating the self-assembly of α-helical PAs.
J. Pept. Sci. 2014; 20: 223–228 Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci
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and caused a tighter array of the peptide segment [45]. The screening of electrostatic repulsion by the metal cations generated an enormous effect on the self-assembly of PAs, resulting in a transition of the morphology of the self-assembled structures. The divalent cation Ca2+ played a greater role to induce the PAs to form tight aggregations compared with the monovalent cation Na+. The CD spectra of PA3 and PA4 in the presence of Na+ and Ca2+ are shown in Figure 6. The α-helical secondary structures of PA3 and PA4 were gradually transformed to β-sheets upon cation (Na+ or Ca2+) addition, revealing a change from the original conformation. A proposed PA self-assembly mechanism is illustrated in Figure 7. Based on previous reports [44,46–48], the driving forces governing the self-assembly of PAs in aqueous medium arise from several combined effects, including hydrophobic interactions of the alkyl tails, steric hindrance effects, electrostatic interactions, and hydrogen bonding among the peptide sequences. The final structural properties such as size and shape reflect a delicate balance of these effects. In PA1 and PA2, hydrophobic interactions between the alkyl tails were the main driving force for the formation of nanofiber structures, and the relatively attenuated hydrophobic interactions of PA3 and PA4 led to the formation of spherical micelles [44]. Increasing the length of the peptides could gradually strengthen the interactions with
MENG ET AL. Acknowledgements This work was supported by the National Natural Science Foundation of China (81202465 and 81373266) and the National Key Technologies R & D Program for New Drugs of China (2012ZX09301003).
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