Barium-triggered β-sheet formation and hydrogelation of a short peptide derivative

Article abstract of DOI:10.1039/c3ra45023f

We reported on a short peptide-taurine conjugate that could adopt β-sheet conformation and form hydrogels triggered by Ba²⁺, which might be applied for the removal of Ba⁺ from water with high Ba ²⁺ content.

Full text of DOI:10.1039/c3ra45023f

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Barium-triggered b-sheet formation and
hydrogelation of a short peptide derivative†
Cite this: RSC Adv., 2014, 4, 1193
a
b
a
a
b
*
Jingjing Mei, Xiaoli Zhang, Meifeng Zhu, Jianing Wang, Ling Wang
a
*
and Lianyong Wang
Received 11th September 2013
Accepted 15th November 2013
DOI: 10.1039/c3ra45023f
www.rsc.org/advances
We reported on a short peptide–taurine conjugate that could adopt causing cardiac irregularities, tremors, weakness, anxiety,
b-sheet conformation and form hydrogels triggered by Ba2+, which dyspnea and paralysis. This may be due to the ability of Ba2+ to
might be applied for the removal of Ba+ from water with high Ba2+ block potassium ion channels which are critical to the proper
content.
function of the nervous system.14 Therefore, the long-term
drinking the water with high Ba2+ content would bring about
serious harm to the human health.
Stimuli-responsive hydrogels of peptides lead to biomaterials
with controllable property and utility for a wide range of
biomedical applications.1 Many peptides or peptide derivatives
can respond rapidly to external stimuli including enzymatic
triggers,2 light irradiation,3 redox,4 addition of polymer and
protein additives,5 addition of salts,6 etc.7 Among these external
stimuli, the addition of metal ion has been of particular
interest. Inspired by the naturally occurring protein–metal and
peptide–metal interactions, several peptides that can form
hydrogels via the addition of metal ions have been rationally
designed and reported. For example, Stupp and co-workers have
reported a calcium-triggered peptide hydrogel system for the
biomineralisation of calcium phosphate.8 Schneider and
co-workers have developed a peptide that can form b-hairpin
structure and hydrogels upon the addition of zinc ion.9 They
have also reported a peptide hydrogel formed by the addition of
heavy metal ions.10 Xu and co-workers have used calcium ions to
trigger the formation of short peptide-based hydrogels and
adjust the elasticity of the resulting hydrogels.11 They have also
reported hydrogel systems responsive to potassium ion and
nickel ion.12 To our knowledge, a barium-triggered peptide
hydrogel system has not been reported.
Stimulated by aforementioned pioneering works, we
hypothesise that the peptide containing sulfonic acid group can
interact with barium ion, resulting in the formation of hydrogel.
To test our hypothesis, we designed and synthesised a dipeptide
molecule bearing taurine, named as Nap-FF-Taurine (Fig. 1).
The synthesis of Nap-FF-Taurine as shown in Fig. S1† was easy
and straightforward. We rstly obtained Nap-FF by solid phase
peptide synthesis. The active N-hydroxylsuccinimide (NHS)
ester of Nap-FF was then used to couple with taurine to yield
Nap-FF-Taurine in a moderate yield of 85%. The pure Nap-FF-
Taurine (Fig. 1) was obtained by reverse phase high perfor-
mance liquid chromatography (HPLC).
We found that the Nap-FF could not well dissolve in PBS
solution due to strong hydrophobic interaction. But the Nap-FF-
Taurine could form homogeneous solutions of in PBS at pH 7.4
when its concentration was lower than 0.7 wt% (Fig. S3†). Aer
the addition of one equiv. of Ba2+ to the solution of Nap-FF-
Taurine. We observed a rapid hydrogel formation (Fig. 1) and
the minimum gelation concentration of Nap-FF-Taurine with
one equiv. of Ba2+ was about 0.2 wt%. For a PBS solution con-
taining 0.2 wt% of Nap-FF-Taurine, the minimum equiv. of Ba2+
to trigger the formation of a gel was about 0.50. However, the
Barium chloride, along with other water-soluble barium
salts, is highly toxic.13 At low doses, barium ions act as a muscle
stimulant, whereas at high doses they affect the nervous system,
aCollege of Life Sciences, Nankai University, Tianjin 300071, P. R. China. E-mail: wly@
nankai.edu.cn; Fax: +86-22-23498775; Tel: +86-22-23502875
bCollege of Pharmacy, Nankai University, Tianjin 300071, P. R. China. E-mail:
chwling@nankai.edu.cn; Fax: +86-22-23498775; Tel: +86-22-23502875
† Electronic supplementary information (ESI) available: Characterization of the
peptide, rheology, 1H NMR, mass spectrum (MS) and uorescence spectra, etc.
See DOI: 10.1039/c3ra45023f
Fig. 1 Chemical structure of the Nap-FF-Taurine and an optical image
of the hydrogel of Nap-FF-Taurine (0.5 wt%) with 1 equiv. of Ba2+
.
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entangled with each other forming a network for hydrogelation.
These results agreed with the observations of rheological
measurements and indicated that addition of barium ions
could lead to bril formation, enhancement of G0 value, and the
formation of hydrogels.
Circular dichroism (CD) spectroscopy is a powerful tool
which provides information about the secondary structure of
the self-assembled peptide in the gel phase.15 So we explored CD
spectroscopy to monitor the triggered folding and self-assembly
of Nap-FF-Taurine in the presence of different equiv. of Ba2+
(Fig. 4A) and different other metal ions (Fig. 4B and C). As
shown in Fig. 4A, Nap-FF-Taurine adopted a random coil
conformation when there was no barium ion. When 0.1 equiv.
of BaCl2 was added to the peptide solution, the b-sheet structure
was observed, indicated by the positive peak at 196 nm and
negative one at 213 nm.15 Moreover, more amounts of Ba2+
would lead to more pronounced peaks, suggesting that the bind
between Ba2+ and Nap-FF-Taurine initiated the transition of
conformation and subsequently peptide assembly into a
b-sheet-rich network. For other metal ions including Zn2+, Sr2+,
Pb2+, Mg2+, Ca2+, Cu2+ and Fe2+, they could not induce b-sheet
formation of Nap-FF-Taurine (Fig. 4B and C), indicating that
there were the weak affinity interaction between these ions and
Nap-FF-Taurine molecules. These observations correlated well
with the results of hydrogel formation test and indicated the
specic interaction between Ba2+ and Nap-FF-Taurine.
To further investigate the molecular arrangement in the
hydrogels, we obtained uorescence emission spectra of
Nap-FF-Taurine in both solution and gel phases. As shown in
Fig. S8,† the emission peaks at 330 nm appeared in both solu-
tion and gel phase. Compared with solution phase, the center of
the emission peaks didn't show red shi in the gel phases,
suggesting that there was no clearly p–p stacking between
naphthalene rings in its gel phase. It was reported that under
proper condition, the Nap-FF molecular have excellent self-
assembly ability to form hydrogels due to p–p interaction.16
However, the conjugation of taurine with Nap-FF would
Fig. 2 (A) Rheology with the mode of dynamic frequency sweep of
solutions of Nap-FF-Taurine with different equiv. of Ba2+ (- no Ba2+
C
0.1 equiv. : 0.5 equiv and ; 0.7 equiv. of Ba2+); (B) the plot of elasticity
(G0) vs. the different equiv. of Ba2+ ([Nap-FF-Taurine] ¼ 0.2 wt%).
addition of one equiv. of other metal ions to the solution of Nap-
FF-Taurine including Fe2+, Ca2+, Pd2+, Mg2+, Sr2+, Cu2+, and Zn2+
could not induce the hydrogel formation (Fig. S3†). These
observations clearly indicated that Ba2+ could selectively induce
the formation of hydrogels of Nap-FF-Taurine and demon-
strated the success of our design.
We rst used a rheometer to study the mechanical properties
of solution of Nap-FF-Taurine and gels formed by treating solu-
tion of Nap-FF-Taurine with different equiv. of Ba2+. As shown in
Fig. 2A, before the addition of barium ions, the solution of Nap-
FF-Taurine possessed the lowest elasticity (G0) value of about 40
Pa at the frequency of 0.1 rad sꢀ1. Upon the addition of 0.1, 0.5,
and 0.7 equiv. of Ba2+ to the solution, the G0 value changed to
bigger one of about 52, 650, 750 Pa, respectively which indicated
that the addition of more Ba2+ would lead to bigger G0 value and
there was a sharp increase in G0 value for the solution with 0.3–
0.6 equiv. of Ba2+ (Fig. 2B). The G0 value of samples with 0.7 and
0.9 equiv. of Ba2+ were similar. These observations correlated
with the fact that 0.5 equiv. of Ba2+ would lead to the hydro-
gelation of solution of Nap-FF-Taurine (0.2 wt%).
To investigate the effect of Ba2+ on the self-assembled
microstructure of the hydrogels, we used transmission electron
microscopy (TEM) to examine the morphology of the solution
and gels. The solution of Nap-FF-Taurine (0.5 wt%) presented a
morphology of irregular aggregates (Fig. 3A). upon the addition
of 0.5 equiv. of Ba2+ to the solution of Nap-FF-Taurine, the
irregular aggregates changed to small and short brils with
diameter of about 35 nm and length shorter than 1.2 mm
(Fig. 3B). For the gel formed by adding 0.7 equiv. of Ba2+, it
exhibited long and uniform bers with the diameter of about 45
nm and length longer than 7.9 mm (Fig. 3C). These brils were
Fig. 3 TEM images of (A) solution of Nap-FF-Taurine (0.5 wt%), (B) gel Fig. 4 Circular dichroism (CD) spectra of solutions of Nap-FF-Taurine
of Nap-FF-Taurine (0.5 wt%) with 0.5 equiv. of Ba2+, and (C) gel of (0.05 wt%) with (A) different equiv. of Ba2+ (B and C) one equiv. of
Nap-FF-Taurine (0.5 wt%) with 0.7 equiv. of Ba2+
.
different metal ions.
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decrease (even completely destroy) the self-assembly ability of 3.31 (m, 3H), d 3.01 (m, 2H), d 2.78 (m, 2H). 13C NMR (DMSO-
Nap-FF due to the increase of exibility of Nap-FF molecule. d6): d 171.1, 170.3, 169.9, 138.0, 137.8, 134.1, 133.0, 131.8, 129.4
Therefore, the resulting conjugate of Nap-FF-Taurine could (2 signals), 129.3 (2 signals), 128.2 (2 signals), 128.0 (2 signals),
form homogeneous solutions in phosphate buffer saline (pH ¼ 127.7, 127.6 (2 signals), 127.5, 127.4, 126.4, 126.2, 126, 125.6,
7.4) solution. Interestingly, the Nap-FF-Taurine can form 54.4, 53.9, 50.4, 42.3, 37.7, 37.5, 35.7 ppm.
hydrogels in the presence of Ba2+ but not in the presence of
Zn2+, Sr2+, Pb2+, Mg2+, Ca2+, Cu2+, and Fe2+. The reason for this
Hydrogel formation
phenomenon might be that the Ba2+ could specic bind with
In order to investigate the hydrogel formation, The Nap-FF-
sulfonic group of Nap-FF-Taurine, leading to form Nap-FF-
Taurine was rst dissolved in a small amount of PBS, then
Taurine–Ba complex which had extremely low solubility similar
sodium carbonate solution were added to adjust the pH to
to the barium sulfate (BaSO4).
about 7.4, aerward, a certain amount of PBS was supple-
We attempted to use Nap-FF-Taurine to remove Ba2+ from
mented to obtain Nap-FF-Taurine solution with desired
water. 3 mg of Nap-FF-Taurine could decrease the concentration
concentration of Nap-FF-Taurine. As shown in Fig. S4,† the
of Ba2+ in 5 mL of water from 850 ppm to 200 ppm, which
indicated that Nap-FF-Taurine could act as potential treatment
agent of water with high Ba2+ content.
gelation occurred aer the addition of 0.5 or 0.7 equiv. of Ba2+
.
However, the gelation didn't occur aer the addition of Ca2+,
Cu2+, Fe2+, Pb2+, Sr2+, Mg2+ and Zn2+ (Fig. S5†).
In summary, we demonstrated that taurine is a useful
functional group for the construction of a peptide conjugate
that could respond to Ba2+. The specic binding of taurine with
barium ion could lead to b-sheet formation of the peptide
conjugate and subsequent hydrogel formation. This is another
example of a hydrogel system that uses metal–ligand coordi-
nation bonding. In addition, this rapid and specic hydro-
gelation system might be applied for the removal of toxic Ba2+
from water with high Ba2+ content. However, a low-cost method
for the peptide's preparation needs to be developed to ensure
economic feasibility, and the structure and components of the
peptide also need to be further optimized so that it can bind
Ba2+ as much as possible.
Removal of Ba2+ from water
To investigate the removal of Ba2+ from water, 5 mL of the
solution containing 850 ppm of Ba2+ was prepared. 3 mg of Nap-
FF-Taurine was then added with nal concentration of 0.06%.
Several minutes later, the occule rather than hydrogel was
formed due to lower concentration than that of gelation. Aer
centrifugalization, the concentration of Ba2+ in supernatant was
determined by Inductively Coupled Plasma-Atomic Emission
Spectrometry (ICP-AES).
Acknowledgements
This work is supported by NSFC (31070856 and 51003049) and
Tianjin MSTC (12JCYBJC11300).
Experiments
Synthesis of Nap-FF-Taurine
Nap-FF was rst synthesised by solid-phase peptide synthesis
(SPPS) methods as described in the ESI.† For synthesis of
Nap-FF-Taurine, the DMF solution containing Nap-FF and NHS
was mixed with aqueous solution of equal amount of Taurine to
form a homogeneous solution in a ask. Aer being cooled to
0 ꢁC in the ice bath, DIEPA was added to adjust the pH to about
8. EDC was then added in the ask. The resulting reaction
mixture was stirred overnight and directly separated by HPLC
with MeOH–water (0.1%) as the eluents and the target product
was collected. As shown in Fig. S1(B),† there is only one narrow
single peak on the LC-MS spectrum indicating the target
product was very pure. The MS data (Fig. S2†) of target product
(MS: (M + 1)+ ¼ 588.69, HR-MS: (M + 1)+ ¼ 588.2165) was very
consistent with that of Nap-FF-Taurine (calc: M+ ¼ 587.69),
proving the pure Nap-FF-Taurine was obtained.
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