Nanostructures from the self-assembly of α-helical peptide amphiphiles

Article abstract of DOI:10.1002/psc.2606

Self-assembly of PAs composed of palmitic acid and several repeated heptad peptide sequences, C₁₅H₃₁CO-(IEEYTKK)_n-NH ₂ (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

Full text of DOI:10.1002/psc.2606

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
on concentration, ionic strength, pH value, temperature, and so
on [25,26]. Stupp and coworkers [27–32] have synthesized a broad
Introduction
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 cationic-
head 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 mono-
alkyl 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 hydro-
phobic 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 con-
fined to the core. PAs can self-assemble into a number of different
structures including micelles, vesicles, and nanofibers, depending
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 hydropho-
bic core and residues at e and g form inter-helical ionic interac-
tions [36]. In this paper, one of the heptad repeat sequences,
*
Correspondence to: Keliang Liu, Beijing Institute of Pharmacology and Toxicology,
Beijing, 100850, China. E-mail: keliangliu55@126.com
†
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.
J. Pept. Sci. 2014; 20: 223–228
Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

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.
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 spec-
trometer (Perkin Elmer Co., Waltham, MA, USA) at an excitation
wavelength of 334 nm, an excitation slit width of 4 nm, and an emis-
sion slit width of 3 nm. The region from 340 to 450 nm was exam-
ined 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.
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.
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 sec-
ondary 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 attrib-
uted 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].
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 spec-
trophotometry (Autoflex III, Bruker Daltonics, Bremen, Germany).
fH ¼ ðθ222–θCÞ=ðθH–θCÞ
θH ¼ ð–4400 þ 250TÞð1–3=NrÞ
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
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.
Transmission Electron Microscopy
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.
spherical micelles and nanovesicles. Thus, the morphology of
Table 1. α-helical contents of PAs and Ac-(IEEYTKK)n-NH2 (n = 3 and 4)
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 rel-
atively 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 proper-
ties 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
Sample name
Peptide sequence
Content of
α-helices (%)
Ac-(IEEYTKK)3-NH2
PA3
Ac-(IEEYTKK)3-NH2
15H31CO-(IEEYTKK)3-NH2
Ac-(IEEYTKK)4-NH2
15H31CO-(IEEYTKK)4-NH2
28.05
36.21
35.13
42.22
C
Ac-(IEEYTKK)4-NH2
PA4
C
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
Figure 3. TEM images of (A) PA1, (B) PA2, (C) PA3, and (D) PA4 in 0.1 mg/ml aqueous mediums.
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.
J. Pept. Sci. 2014; 20: 223–228 Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

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
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
Table 2. CMC values of PAs
Sample name
Peptide sequence
CMC(μM)
PA1
PA2
PA3
PA4
C15H31CO-(IEEYTKK)1-NH2
C15H31CO-(IEEYTKK)2-NH2
C15H31CO-(IEEYTKK)3-NH2
C15H31CO-(IEEYTKK)4-NH2
5.62
6.76
10.14
11.17
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.
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.
and caused a tighter array of the peptide segment [45]. The
screening of electrostatic repulsion by the metal cations gener-
ated 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 interac-
tions of the alkyl tails, steric hindrance effects, electrostatic inter-
actions, 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
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

MENG ET AL.
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Acknowledgements
This work was supported by the National Natural Science Founda-
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