Self-assembly of amphiphilic peptides into bio-functionalized nanotubes: A novel hydrolase model

Article abstract of DOI:10.1039/c3tb20156b

Herein, we report the construction of a novel hydrolase model via self-assembly of a synthetic amphiphilic short peptide (Fmoc-FFH-CONH ₂) into nanotubes. The peptide-based self-assembled nanotubes (PepNTs-His) with imidazolyl groups as the catalytic centers exhibit high catalytic activity for p-nitrophenyl acetate (PNPA) hydrolysis. By replacement of the histidine of Fmoc-FFH-CONH₂ with arginine to produce a structurally similar peptide Fmoc-FFR-CONH₂, guanidyl groups can be presented in the nanotubes through the co-assembly of these two molecules to stabilize the transition state of the hydrolytic reaction. Therefore significantly improved catalytic activity has been achieved by the reasonable distribution of three dominating catalytic factors: catalytic center, binding site and transition state stabilization to the co-assembled peptide nanotubes (PepNTs-His-Arg_max). The resulting hydrolase model shows typical saturation kinetics behaviour to that of natural enzymes and the catalytic efficiency of a single catalytic center is 519-fold higher than that without catalysts. As for a nanotube with multi-catalytic centers, a remarkable catalytic efficiency could be achieved with the increase of building blocks. This model suggests that the well ordered and dynamic supramolecular structure is an attractive platform to develop new artificial enzymes to enhance the catalytic activity. Besides, this novel peptide-based material has excellent biocompatibility with human cells and is expected to be applied to organisms as a substitute for natural hydrolases. The Royal Society of Chemistry.

Full text of DOI:10.1039/c3tb20156b

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Materials Chemistry B
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PAPER
Self-assembly of amphiphilic peptides into bio-
functionalized nanotubes: a novel hydrolase model
Cite this: J. Mater. Chem. B, 2013, 1,
2297
Zupeng Huang, Shuwen Guan, Yongguo Wang, Guannan Shi, Lina Cao, Yuzhou Gao,
Zeyuan Dong, Jiayun Xu, Quan Luo and Junqiu Liu
*
*
Herein, we report the construction of a novel hydrolase model via self-assembly of a synthetic amphiphilic
short peptide (Fmoc-FFH-CONH2) into nanotubes. The peptide-based self-assembled nanotubes (PepNTs-
His) with imidazolyl groups as the catalytic centers exhibit high catalytic activity for p-nitrophenyl
acetate (PNPA) hydrolysis. By replacement of the histidine of Fmoc-FFH-CONH2 with arginine to produce
a structurally similar peptide Fmoc-FFR-CONH2, guanidyl groups can be presented in the nanotubes
through the co-assembly of these two molecules to stabilize the transition state of the hydrolytic
reaction. Therefore significantly improved catalytic activity has been achieved by the reasonable
distribution of three dominating catalytic factors: catalytic center, binding site and transition state
stabilization to the co-assembled peptide nanotubes (PepNTs-His-Argmax). The resulting hydrolase model
shows typical saturation kinetics behaviour to that of natural enzymes and the catalytic efficiency of a
single catalytic center is 519-fold higher than that without catalysts. As for a nanotube with multi-
catalytic centers, a remarkable catalytic efficiency could be achieved with the increase of building blocks.
This model suggests that the well ordered and dynamic supramolecular structure is an attractive
platform to develop new artificial enzymes to enhance the catalytic activity. Besides, this novel peptide-
based material has excellent biocompatibility with human cells and is expected to be applied to
organisms as a substitute for natural hydrolases.
Received 1st February 2013
Accepted 5th March 2013
DOI: 10.1039/c3tb20156b
www.rsc.org/MaterialsB
Although the strategies mentioned above are effective to
simulate enzymes with extremely high activity and other excel-
Introduction
Natural enzymes are a type of highly efficient biocatalyst.
Compared to common chemical catalysts, they have many
advantages, and can catalyze reactions with remarkable cata-
lytic activity and specicity under mild conditions. However,
natural enzymes are proteins, RNAs and their complexes which
are difficult to acquire, easily denatured and deactivated under
various physical or chemical factors, and for these reasons
most of them are extraordinarily expensive. Therefore, an
understanding of the underlying catalytic principles, struc-
ture–function relationships and how to design and synthesize
articial enzymes that are easily prepared, stable, cheap and
exhibit similar or higher catalytic activities than natural
enzymes based on these principles are of great interest for
many scientists.
lent properties, they are unable to achieve the reasonable match
among the catalytic factors of enzymes due to the lack of
dynamic ordered structures caused by their stable covalent
interactions. To address this problem, a new strategy of supra-
molecular self-assembly for enzymatic mimicking has been
developed recently. The advantages of this strategy are that it is
simple, low-cost, dynamic and efficiently generates highly
ordered structures with complex architectures to construct
tunable articial enzymes.10 Since the driving forces of self-
assembly are non-covalent interactions like hydrogen bonds,
electrostatic interactions, hydrophobic interactions, p–p
stacking and so on, enzyme mimics constructed based on this
strategy provide several features analogous to natural enzymes:
(i) dynamic supramolecular structures; (ii) amphiphilicity of
supramolecular architectures; (iii) supramolecular interactions
as driving forces for substrate recognition; (iv) specic and/or
hydrophobic microenvironments for catalysis.11 Therefore, the
self-assembly strategy is expected to be an alternative way for
simulating the structures and functions, and investigating the
structure–function relationships and the catalytic mechanisms
of natural enzymes.
In the past several decades, considerable efforts have been
made to employ various strategies to mimic natural enzymes,
such as organic synthesis,1–4 semi-synthesis,5 molecular
imprinting,6,7 genetic engineering and so on.8,9
State Key Laboratory of Supramolecular Structure and Materials, College of
Chemistry, Jilin University, Changchun 130012, People's Republic of China. E-mail:
junqiuliu@jlu.edu.cn; luoquan@jlu.edu.cn; Fax: +86-431-85193421
Among various self-assembled systems, the self-assembly of
peptide-based molecules into well ordered nanostructures12–27
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has aroused great interest for biochemists. Since bioactive
peptides are easily synthesized, engineered, chemically modi-
ed and biologically functionalized, they are oen chosen as
suitable building blocks to fabricate nanostructured materials
that have many biotechnological applications.28–34 Reches and
Gazit rst reported that diphenylalanine dipeptides can self-
assemble into nanotubes in water which can be used to
produce discrete silver nanowires.35 Subsequently, Li's group
observed that the nanotubes formed from cationic diphenyla-
lanine dipeptide can be transformed into vesicles upon dilu-
tion which were used to deliver oligonucleotide into cells.24
Lately, Stupp and Guler reported a nanober hydrolytic cata-
lyst36 with highly ordered interior constructed from peptide
amphiphiles (PAs) which exhibits considerable enhancement
in catalytic rate. Up to now a few cases have been reported to
use amphiphilic peptide amphiphiles for fabricating articial
enzymes; however, nanotube hydrolase models formed by
co-assembly of amphiphilic short peptides have not been
reported yet.
Given the fact that the imidazolyl group of histidine plays
an important role in the catalytic process of native hydrolase
(e.g.: chymotrypsin,37 trypsin,38 acetylcholine esterase,39 and
kidney dialkyluorophosphatase40), herein we designed and
synthesized an imidazolyl-containing amphiphilic tripeptide
N-(uorenyl-9-methoxycarbonyl)-Phe-Phe-His-NH2 (Fmoc-FFH-
CONH2) hoping to exploit it to construct a novel supramolec-
ular peptide hydrolase model with high catalytic activity, good
biocompatibility and based on it to study the relationships
between the ordered arrangement of the supramolecular
structures and the catalytic activity. We use peptides as scaf-
folds because the exible structures of peptides do make it
possible to put the special functional residues of complex
proteins into simple molecules, which could further self-
assemble into supramolecular structures with abundant
orderly arranged functional groups that have many unexpected
properties. Moreover, such peptide-assembled structures are
also considered low-toxic and biocompatible, and are expected
to be applied in organisms as substitutes for natural hydro-
lases because all constituent units of peptides are derived from
natural amino acids. As expected, the synthetic tripeptide can
self-assemble into uniform ultra-thin nanotubes with high
catalytic activity for p-nitrophenyl acetate (PNPA) hydrolysis. In
order to investigate if the incorporation of binding sites for
substrate PNPA could contribute to the improvement in cata-
lytic activity, different proportions of guanidyl groups in Fmoc-
FFR-CONH2 were incorporated into the nanotubes as stable
transition state binding sites via the co-assembly method.
Optimizing the articial hydrolase enzyme models was ach-
ieved through changing the molar ratio of catalytic centers
(Fmoc-FFH-CONH2) and binding sites (Fmoc-FFR-CONH2).
Materials and methods
Materials
Fmoc-protected amino acids (Fmoc-Phe-OH, Fomc-His(Trt)-OH
and Fmoc-Arg(Pbf)-OH), anhydrous N-hydroxybenzotriazole
(HOBt anhydrous), N-methylmorpholine (NMM), benzotriazol-1-yl-
oxytripyrrolidinophosphonium hexauorophosphate (PyBOP) and
Rinkamide-AMresinwerepurchasedfromGLBiochem(Shanghai)
Ltd. and used as received. p-Nitrophenyl acetate (PNPA) was
purchased from J&K Chemical Ltd. (Beijing, China). Other
reagents and solvents were purchased from Sigma and used as
supplied. Dichloromethane (DCM) and dimethyl formamide
(DMF) were purchased commercially, redistilled and subse-
quently dried with molecular sieves prior to use. All water used
was from a Millipore water purication system with a minimum
resistivity of 18.0 MU cm.
Synthesis and purication of Fmoc-FFH-CONH2 (Scheme 1)
The synthesis and purication of N-terminally Fmoc-protected
peptides, Fmoc-FFH-CONH2 and Fmoc-FFR-CONH2, were per-
formed using a similar method to that reported previously.28
Normally, Fmoc-FFH-CONH2 (Fmoc-Phe-Phe-His-CONH2) was
constructed on a preloaded Rink amide-AM resin using stan-
dard Fmoc (9-uorenylmethoxycarbonyl) solid peptide phase
synthesis techniques on a 0.5 mmol scale. All amide-coupling
reactions of peptide synthesis were accomplished for 1.5 hours
using 4 equiv. of each amino acid, 4 equiv. of PyBOP, 4 equiv. of
HOBt, and 4 equiv. of NMM in DMF. Fmoc removals were
carried out with 30 min treatments of 20% (by volume) piperi-
dine–DMF solution. Cleavage of the peptides from the resin was
performed with a mixture of triuoroacetic acid (TFA)–H2O–
triisopropylsilane (95 : 2.5 : 2.5 by volume) for 3 h. The crude
peptide product was precipitated and then washed with cold
diethyl ether at least four times. The resulting white product
was cryo-dried and puried by reversed-phased HPLC on a
preparative Varian Dynamax C18 column under linear elution
gradients of CH3CN–water (30/70, 0.1% TFA) over 30 minutes.
Peptide purity was assessed by analytical reversed-phase HPLC
(>98%) and characterized using a Bruker 300 MHz spectrometer
and matrix assisted laser desorption ionization-time of ight
mass spectrometer (MALDI-TOF MS).
1
ꢀ
H NMR (300 MHz, DMSO-d6, 25 C) d (ppm): 8.85 (s, 1H),
8.34 (d, 1H), 8.26 (d, 1H), 7.88 (d, 2H), 7.59 (t, 2H), 7.53 (d, 1H),
7.40 (t, 3H), 7.24 (m, 15H), 4.49 (s, 2H), 4.06–4.27 (m, 5H), 2.53–
3.13 (m, 5H). C39H38N6O5 m/z (ES) 693.2 [M + Na]+.
Then a co-assembled hydrolase model (PepNTs-His-Argmax
)
that is more efficient than PepNTs-His was obtained. Subse-
quently, in this report, atomic force microscopic (AFM)
imaging was exploited to discuss the relationships between the
ordered structure and catalytic activity in detail and an MTT
assay was performed to test the cytotoxicity of this peptide-
based articial enzyme.
Scheme 1 The structure of Fmoc-FFH-CONH2.
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diluting the stock solution with ultra-pure water to a nal Fmoc-
FFH-CONH2 concentration of 1 mM with pH ¼ 7.5.
Characterization of the co-assembly behaviour
Fmoc-FFH-CONH2 and Fmoc-FFR-CONH2 at a molar ratio of
1 : 1 were mixed in deuterated DMSO (DMSO-d6) at a total
concentration of 100 mM. The stock solution was then diluted
with deuteroxide to a nal concentration of 2 mM. Individual
1 mM Fmoc-FFH-CONH2 and Fmoc-FFR-CONH2 aqueous solu-
tions were also prepared as controls. The fresh samples were
characterized by 1H NMR spectroscopy. Changes of peaks on
Fmoc groups, benzene rings and imidazolyls before and aer
co-assembly were compared to identify if these two molecules
could co-assemble.
Scheme 2 The structure of Fmoc-FFR-CONH2.
Synthesis and purication of Fmoc-FFR-CONH2 (Scheme 2)
The synthesis and purication of Fmoc-FFR-CONH2 were similar
to those of Fmoc-FFH-CONH2 with the same amide-coupling,
Fmoc removal and purication steps except that cleavage of the
peptides from the resin was performed with a mixture of tri-
uoroacetic acid (TFA)–thioanisole–H2O–phenol–1,2-ethanedi-
thiol (EDT)–triisopropylsilane (81.5 : 5 : 5 : 5 : 2.5 : 1 by volume).
The puried peptide was characterized using a Bruker 300 MHz
spectrometer and matrix assisted laser desorption ionization-time
of ight mass spectrometer (MALDI-TOF MS).
H NMR (300 MHz, DMSO-d6, 25 C) d (ppm): 8.14 (2H, m),
7.89 (2H, d), 7.60 (3H, m), 7.48 (1H, t), 7.40 (3H, m), 7.22 (15H,
m), 4.57 (1H, m), 4.20 (2H, m), 4.10 (2H, m), 3.08 (3H, m), 2.89
(2H, d), 2.72 (1H, m), 1.68 (1H, m), 1.48 (3H, m). C39H43N7O5 m/z
(ES) 712.4 [M + Na]+.
Determination of the catalytic activity for PNPA hydrolysis
The catalytic activity was monitored using PNPA as substrate
ꢀ
(see Fig. 1). The reaction was carried out at 25 C in 500 mL of
N-(2-hydroxyethyl)piperazine-N0-ethanesulfonic acid (HEPES)
buffer (pH ¼ 7.5, 10 mM) containing 0.1 mM of peptide nano-
tube catalyst and 0.5 mM of PNPA. Considering the UV spec-
trum of the hydrolytic product 4-nitrophenol which exhibits a
strong absorption peak at 400 nm, the initial reaction rates were
determined by monitoring the absorption increase at 400 nm
with a Shimadzu 2450 UV-VIS-NIR spectrophotometer. The
appropriate control activity without peptide nanotube catalyst
was also assayed and subtracted from the catalyzed reaction.
The detailed process occurred as follows: rst, in a 0.5 mL
quartz cuvette, 400 mL of HEPES buffer (pH 7.5, 10 mM) and
50 mL of the peptide nanotube catalyst (1 mM) were added and
thermally equilibrated for 2 min. The reaction was initiated by
addition of 50 mL substrate PNPA solution (0.5 mM). The
absorption increase at l ¼ 400 nm due to the release of product
4-nitrophenol was recorded.
1
ꢀ
Preparation of self-assembled peptide nanotube hydrolase
model PepNTs-His
Fmoc-FFH-CONH2 was dissolved in dimethyl sulfoxide (DMSO)
at a concentration of 50 mM. The self-assembled peptide
nanotubes (PepNTs-His) were prepared by diluting the stock
solution with ultra-pure water to a nal peptide concentration
of 1 mM with pH ¼ 7.5.
Cell culture
HeLa cells (epithelial cervical cancer cell line) in Dulbecco's
modied Eagle medium (Invitrogen, Carlsbad, CA) were added
to each well of a 96-well plate supplemented with 10% fetal
bovine serum (FBS), 100 U mLꢁ1 penicillin, and 100 mg mLꢁ1
streptomycin. The cells were grown in a humidied incubator
(37 ꢀC, 5% CO2) and seeded at a density of 1 ꢂ 105 cells per well.
Aer incubation for 12 h, the cells of ꢃ80% conuence were
further incubated with fresh medium containing PepNTs-His or
PepNTs-His-Argmax with set concentrations (from 1 to 100 mM)
for another 24 h. Then 10 mL of 5 mg mLꢁ1 thiazolyl blue
tetrazolium bromide (MTT, Sigma-Aldrich, USA) solution was
Preparation of co-assembled peptide nanotube hydrolase
models PepNTs-His-Args
Different proportions of Fmoc-FFH-CONH2 and Fmoc-FFR-
CONH2 were mixed in dimethyl sulfoxide (DMSO) with a xed
concentration of 50 mM of Fmoc-FFH-CONH2. The co-assem-
bled peptide nanotubes (PepNTs-His-Arg) were prepared by
ꢀ
added. Aer incubation at 37 C for 4 h. DMSO (Sigma, USA)
solution was added to each well to lyse the cells. The absorbance
of supernatants was measured at 570 nm by using a microplate
reader (Bio-Rad ELX800, USA).
Instruments and measurement
Atomic force microscopy (AFM), transmission electron micros-
copy (TEM), circular dichroism (CD), Fourier transform infrared
Fig. 1 The hydrolysis process of PNPA in the presence of enzyme.
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Fig. 2 (A) Height AFM image of nanotubes self-assembled from Fmoc-FFH-
CONH2 (PepNTs-His) in aqueous solution at concentration of 1 mM. (B) TEM
micrograph and HR-TEM micrograph (inset, scale bar is 10 nm) of PepNTs-His,
which was stained with 2% phosphotungstic acid before imaging.
Fig. 4 Fluorescence emission spectra of Fmoc-FFH-CONH2 peptide nanotubes
(PepNTs-His) at concentrations of 0.01 mM (a) and 0.1 mM (b).
(FTIR) spectroscopy, uorescence emission spectroscopy and
UV-vis spectroscopy measurements were performed to charac-
terize the structural conformations and morphologies of
PepNTs-His and PepNTs-His-Arg. The CD spectra were collected
on a Jasco J-810 spectropolarimeter under a nitrogen atmo-
sphere. AFM imaging was performed on a NanoScope Multi-
Mode-AFM (Veeco, USA). TEM measurements were performed
on a JEOL model JEM-3010 at 300 kV. All samples were stained
with 2% phosphotungstic acid. FT-IR spectra were obtained on
a Bruker Vector 22 FT-IR spectrometer at ambient temperature.
UV-vis spectra were measured on a Shimadzu 3100 UV-vis-NIR
spectrophotometer. Fluorescence emission spectra were recor-
ded on a Perkin-Elmer LS-55 luminance spectrometer. 1H NMR
16 nm, inner diameters between 6 and 8 nm and lengths up to
several micrometers. This is consistent with the AFM results.
Dynamic light scattering (DLS) was further utilized to study the
aggregation behavior of the Fmoc-peptides in aqueous solution.
The results (>100 nm) reveal that the Fmoc-peptides can form a
nano-scale structure in aqueous solution through self-assembly
into uniform nanotubes.
Characterization of the secondary structure of the peptide
nanotubes
Circular dichroism (CD) and Fourier-transform infrared (FT-IR)
spectroscopy were employed to study the backbone orientation
of Fmoc-FFH-CONH2 in the nanotubes. A negative peak at 218
nm in the CD spectrum was detected, indicating the presence of
b-sheet structures in the molecular assembly (Fig. 3A).30 This
assertion was further supported by FT-IR spectroscopy study
(Fig. 3B). The FT-IR spectrum of Fmoc-FFH-CONH2 nanotubes
spectra were obtained using
apparatus.
a Bruker AVANCE III 500
Results
Characterization of the self-assembled nanotubes
exhibited absorption peaks at around 1640 and 1690 cmꢁ1
,
AFM images (Fig. 2A) clearly show the formation of uniform,
high aspect ratio cylindrical nanostructures with diameters which are consistent with a b-sheet structure and an anti-
about 10 nm and length of several micrometers. The dense parallel arrangement of the peptides.30,41
nanotubes were intertwined into a net structure, which was
In order to comprehensively investigate the self-assembly
further supported by the hydrogelation of Fmoc-FFH-CONH2 at mechanism, uorescence emission spectroscopy was employed
a higher concentration. Transmission electron microscopy to explore the internal microenvironment among the uorenyl
(TEM) has also been performed to investigate the morphology (Fmoc) moieties of Fmoc-FFH-CONH2 (Fig. 4). At a low
of the peptide nanotubes. TEM images aer negative staining
(Fig. 2B) also obviously revealed the generation of cylindrical
nanotubes with dark walls and relatively off-white hollows.
These nanotubes have external diameters between 14 and
Fig. 3 (A) Circular dichroism spectrum and (B) FT-IR spectrum of Fmoc-FFH-
CONH2 peptide nanotubes (PepNTs-His) at a concentration of 0.1 mM.
Fig. 5 Circular dichroism spectra of PepNTs-His (a) and PepNTs-His-Argmax (b).
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Fig. 7 Plots of absorbance vs. time for hydrolysis of PNPA (0.5 mM). (a) Control,
(b) PepNTs-His, (c) PepNTs-His-Argmax
.
Fig. 6 1H NMR spectra of Fmoc-FFH-CONH2 (a), Fmoc-FFR-CONH2 (b) and their
mixture (c) in D2O.
and cleaved (Fig. 6c). The fact that the observed broad peak of
the mixture is not merely a superposition of individual peaks of
the two components strongly supports the formation of the
mixed nanotubes in which two molecules form a single aggre-
gate structure (PepNTs-His-Arg). The broad peak of Fmoc
groups and benzene rings also reveals that the Fmoc groups and
benzene rings interacted with each other which further
conrms the assertion mentioned above that an extensive
J-aggregate of phenyl rings as well as uorenyl rings exists in the
nanotubes.
concentration of 0.01 mM, PepNTs-His exhibits a sharp emis-
sion peak at 311 nm. As the concentration increases to 0.1 mM,
the emission peak shis to 318 nm. The red-shi is caused by
the interaction of the Fmoc groups of Fmoc-FFH-CONH2 during
the self-assembly process,42,43 which also indicates an anti-
parallel arrangement of uorenyl rings.30 A broad feature with a
maximum at 432 nm in the emission spectrum of Fmoc-FFH-
CONH2 was detected, implying an extensive J-aggregate of
phenyl rings as well as uorenyl rings.30 Similar aggregates were
reported previously in other systems.44 The analytical results
from uorescence spectra indicate that p–p stacking interac-
tions assisted by hydrogen bonds can act as important driving
forces in Fmoc-peptide assembly and the Fmoc-peptide
assembly predominantly aggregates in an anti-parallel b-sheet
manner.
CD measurement was employed to investigate whether the
addition of small amounts of Fmoc-FFR-CONH2 to Fmoc-FFH-
CONH2 would change the secondary structure of the peptide
nanotubes. The comparison of the CD spectra of PepNTs-His
and PepNTs-His-Arg showed that both types of nanotubes
exhibit extremely similar negative peaks at 218 nm (Fig. 5),
which means that the incorporation of small amounts of Fmoc-
FFR-CONH2 into Fmoc-FFH-CONH2 nanotubes does not inter-
rupt the inner secondary structure. Namely, these two types of
nanotubes have the same b-sheet arrangement and aggregate
behavior.
Catalytic behaviour
The initial hydrolytic rate was measured according to the
increased absorption value of the hydrolytic product p-nitro-
phenol at 400 nm. The hydrolytic rate against PNPA without
enyzme is very slow (Fig. 7a). The addition of PepNTs-His results
in a distinct enhancement (Fig. 7b). When Fmoc-FFR-CONH2
was incorporated into the PepNTs-His nanotubes as the binding
site of the stable transition state, under identical conditions, a
higher hydrolytic rate was observed (Fig. 7c) which means the
co-assembled peptide nanotube hydrolase model is more
effective than the self-assembled one. This results implies that
the catalytic activity of enzyme models can be improved by
appropriately strengthening the binding of the stable transition
state.
Characterization of the co-assembly of PepNTs-His-Arg
1H NMR spectroscopy was performed in D2O solutions made up
of Fmoc-FFH-CONH2, Fmoc-FFR-CONH2 and their mixture to
investigate the co-assembly of these two molecules. Represen-
tative relatively sharp peaks of Fmoc groups and benzene rings
were observed in both cases of individual Fmoc-FFH-CONH2
(Fig. 6a) and Fmoc-FFR-CONH2 (Fig. 6b). When they were
mixed, the sharp peaks disappear and a broad peak appears
(Fig. 6c). What's more, aer mixing, the original obvious peaks
Fig. 8 Plots of catalytic reaction rates for hydrolysis of PNPA (0.5 mM) vs. molar
ratio of Fmoc-FFH-CONH2 to Fmoc-FFR-CONH2. The concentration of Fmoc-FFH-
(Fig. 6a) on the imidazolyls of Fmoc-FFH-CONH2 were shied CONH2 was fixed at 0.1 mM.
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Fig. 10 Lineweaver–Burk plots for the hydrolysis of PNPA catalyzed by PepNTs-
His-Argmax (0.1 mM catalytic center Fmoc-FFH-CONH2, Fmoc-FFH-CONH2 : Fomc-
FFR-CONH2 ¼ 20 : 1).
stabilized with the increase of Fmoc-FFR-CONH2. However,
once the proportion of Fmoc-FFH-CONH2 to Fmoc-FFR-CONH2
is below 20 : 1, then the catalytic activity decreases. This is
mainly due to the damaged nanotube scaffolds inuencing the
reasonable distribution of the catalytic centers and binding
sites. On the other hand, when the amount of Fmoc-FFR-
CONH2 is large enough and the substrate-binding ability
endowed from Fmoc-FFR-CONH2 increases to a higher degree,
the excessively strongly binding of substrates will prevent the
products from moving away from the catalytic centers, thus
making the completion of the catalytic cycle inefficient,45–47 and
resulting in lower catalytic activity. At a molar ratio of 20 : 1, the
nanotube structure remains stable and the catalytic centers and
binding sites match each other well, leading to the maximum
catalytic activity and the optimal peptide nanotube hydrolase
model.
Fig. 9 Height AFM images of nanotubes induced by the molar ratio of Fmoc-
FFH-CONH2 to Fmoc-FFR-CONH2: (A) 40 : 1; (B) 20 : 1; (C) 10 : 1; (D) 2 : 1.
Optimization of the co-assembled hydrolase model
To determine whether the incorporation of Fmoc-FFR-CONH2
can achieve higher catalytic activity, a series of co-assembled
peptide nanotubes were prepared by optimizing the molar ratio
between Fmoc-FFH-CONH2 and Fmoc-FFR-CONH2. In this
experimental process, the concentration of Fmoc-FFH-CONH2
was kept constant at 1 mM while the concentration of Fmoc-
FFR-CONH2 was varied. The catalytic activities of different co-
assembled nanotube models were measured under the same
conditions. As shown in Fig. 8, the initial hydrolytic rates of co-
assembled nanotube models can be divided into two stages
according to the increase of molar ratio of these two building
blocks (Fmoc-FFH-CONH2–Fmoc-FFR-CONH2). From 40 : 1 to
20 : 1, the initial rate increases with the increase of Fmoc-FFR-
CONH2. Then the initial hydrolytic rate decreases even though
the molar ratio keeps increasing from 20 : 1 to 2 : 1, revealing
that the initial hydrolytic rate is optimal at the molar ratio
of 20 : 1.
Enzymatic behaviour
Hydrolytic reaction was carried out at all individual concentra-
tions of PNPA to investigate the enzymatic properties of the co-
assembled nanotube model (Fig. 10). A typical enzymatic satu-
ration kinetics (Fig. 11) was measured which proved it to be a
real enzyme model for ester hydrolysis. The apparent kinetic
parameters were calculated based on the Michaelis–Menten
equation: kcat(app) ¼ 1.38 ꢂ 10ꢁ3 sꢁ1, Km ¼ 0.76 mM, kcat(app)
/
AFM images were used to explore the details that cause this
interesting phenomenon. As shown in Fig. 9, from 40 : 1 to
20 : 1, just a small amount of Fmoc-FFR-CONH2 was incorpo-
rated into the nanotubes, and there was no obvious structural
change observed. The co-assembled peptide nanotubes have
similar shapes as the self-assembled peptide nanotubes.
However, as the molar ratio of Fmoc-FFH-CONH2–Fmoc-FFR-
CONH2 decreased below 20 : 1, the nanotube structures were
gradually damaged. It is suggested that the well ordered
supramolecular structure of the peptide nanotubes plays an
important role in their catalytic activity. A similar phenomenon
had also been described by Stupp and Guler.36 At high propor-
tion of Fmoc-FFH-CONH2 to Fmoc-FFR-CONH2, the greater the
amount of Fmoc-FFR-CONH2, the higher the catalytic activity.
Fig. 11 Plot of initial catalytic reaction rate vs. concentration of PNPA in the
presence of PepNTs-His-Argmax (0.1 mM catalytic center Fmoc-FFH-CONH2, Fmoc-
This is mainly due to the transition state being gradually FFH-CONH2 : Fomc-FFR-CONH2 ¼ 20 : 1).
2302 | J. Mater. Chem. B, 2013, 1, 2297–2304
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Journal of Materials Chemistry B
nanotubes to 100 mM (67 mg mLꢁ1), it displayed only a mild
decrease in human cell viability, which obviously suggested that
the peptide-based nanotubes have reasonably good biocom-
patibility with human cells.
Proposed catalytic mechanism
Scheme 3 graphically describes the possible catalytic mechanism
of the co-assembled peptide nanotube hydrolase model. First, the
hydrophobic interior of the peptide nanotubes can help to bind
the benzene ring of aromatic substrate PNPA. Second, an imi-
dazolyl of histidine residue binds a proton of H2O molecule to
activate the hydroxide ion. The hydroxide ion, as a nucleophilic
moiety, further attacks the carbonyl of PNPA to generate a
tetrahedral transition state. Finally, a guanidyl on the co-assem-
bled nanotubes binds the oxide ions of the tetrahedral interme-
diate complex to stabilize the tetrahedral transition state and
activates the C–O bond for its cleavage. Product p-nitrophenol is
then released and the next catalysis cycle begins.
Fig. 12 Cytotoxicity of PepNTs-His and PepNTs-His-Argmax at different concen-
trations. The viability of HeLa cells incubated with different concentrations (mM) of
PepNTs-His for 24 h.
Km ¼ 1.82 Mꢁ1 sꢁ1, kcat(app)/kuncat ¼ 519. This means a single
catalytic center of the nanotubes exhibits 519-fold higher cata-
lytic activity for PNPA hydrolysis compared to that without
Conclusions
catalyst. Given a nanotube with multi-catalytic centers, supra- In conclusion, a novel nanotube hydrolase model self-assem-
molecular nanoenzymes would exhibit more remarkable bled from synthetic amphiphilic short peptide Fmoc-FFH-
catalytic activity.
CONH2 has been prepared. Taking advantage of the hydro-
phobic microenvironment provided by the Fmoc-FF aromatic
ring segments and the catalytic center histidines loaded on the
self-assembled peptide nanotubes, this model exhibits higher
catalytic activity for PNPA hydrolysis compared to that without
catalyst. Then Fmoc-FFR-CONH2 which contains guanidyls,
the binding groups for carbonyls, was incorporated into the
Fmoc-FFH-CONH2 nanotubes to form a co-assembled peptide
nanotube hydrolase model. At a suitable molar ratio of Fmoc-
FFH-CONH2–Fmoc-FFR-CONH2, the co-assembled model
exhibits the highest catalytic ability. A comparison of the cata-
lytic activity between PepNTs-His and PepNTs-His-Arg
combined with the AFM results reveals that both a well-ordered
dynamic supramolecular structures and the reasonable match
between the catalytic centers and binding sites play important
roles in the improvement of the catalytic activity of articial
enzymes. More importantly, this novel peptide hydrolase model
is biocompatible with human cells which is promising for use
as substitutes for natural hydrolases to treat inammation,
anorexia, edema and other diseases.
Cytotoxicity study of PepNTs-His and PepNTs-His-Argmax
An MTT assay was performed to evaluate the cytotoxicity of
PepNTs-His and PepNTs-His-Argmax. The effect of the nano-
tubes on the proliferation of HeLa was studied (Fig. 12). Aer
24 h of incubation, a slight decrease in cell viability was
observed. Even increasing the concentration of the peptide
Acknowledgements
We are grateful for the nancial support from the Natural
Science Foundation of China (no: 21234004, 91027023,
21221063, 21004028), Changchun Institute of Applied Chem-
istry (RERU2011008) and the Ministry of Education of China.
Notes and references
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Scheme
3
Schematic representation of the possible mechanism for the
cleavage of PNPA catalyzed by PepNTs-His-Argmax
.
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