Salt-induced control of supramolecular order in biocatalytic hydrogelation

Article abstract of DOI:10.1021/la303388s

Biocatalytic action and specific ion effects are both known to have dramatic effects on molecular self-assembly and hydrogelation. In this paper, we demonstrate that these effects are highly cooperative. Biocatalytic hydrogelation of Fmoc peptides in the presence of salts combines kinetic (through enzymatic catalysis) and thermodynamic (specific ion and protein templating) contributions when applied in combination. Spectroscopic data (obtained by fluorescence spectroscopy and circular dichroism) revealed that hydrophobic interactions are greatly affected, giving rise to differential chiral organization and supramolecular structure formation. The kinetic effects of catalytic action could be removed from the system by applying a heat/cool cycle, giving insight into the thermodynamic influence of both protein and salt on these systems and showing that the effects of catalysis, templating, and salts are cooperative. The variable molecular interactions are expressed as variable material properties, such as thermal stability and mechanical strength of the final gel-phase material. To gain more insight into the role of the enzyme, beyond catalysis, in the underlying mechanism, static light scattering is performed, which indicates the different mode of aggregation of the enzyme molecules in the presence of different salts in aqueous solution that may play a role to direct the assembly via templating. Overall, the results show that the combination of specific salts and enzymatic hydrogelation can give rise to complex self-assembly behaviors that may be exploited to tune hydrogel properties.

Full text of DOI:10.1021/la303388s

Article
pubs.acs.org/Langmuir
Salt-Induced Control of Supramolecular Order in Biocatalytic
Hydrogelation
Sangita Roy,*,† Nadeem Javid,† Jan Sefcik,‡ Peter J. Halling,† and Rein V. Ulijn*,†
†WestCHEM/Department of Pure and Applied Chemistry, University of Strathclyde, 295 Cathedral Street, Glasgow, G1 1XL, United
Kingdom
‡Department of Chemical and Process Engineering, University of Strathclyde, 75 Montrose Street, Glasgow G1 1XJ, United Kingdom
S
* Supporting Information
ABSTRACT: Biocatalytic action and specific ion effects are
both known to have dramatic effects on molecular self-
assembly and hydrogelation. In this paper, we demonstrate
that these effects are highly cooperative. Biocatalytic hydro-
gelation of Fmoc peptides in the presence of salts combines
kinetic (through enzymatic catalysis) and thermodynamic
(specific ion and protein templating) contributions when
applied in combination. Spectroscopic data (obtained by
fluorescence spectroscopy and circular dichroism) revealed
that hydrophobic interactions are greatly affected, giving rise to differential chiral organization and supramolecular structure
formation. The kinetic effects of catalytic action could be removed from the system by applying a heat/cool cycle, giving insight
into the thermodynamic influence of both protein and salt on these systems and showing that the effects of catalysis, templating,
and salts are cooperative. The variable molecular interactions are expressed as variable material properties, such as thermal
stability and mechanical strength of the final gel-phase material. To gain more insight into the role of the enzyme, beyond
catalysis, in the underlying mechanism, static light scattering is performed, which indicates the different mode of aggregation of
the enzyme molecules in the presence of different salts in aqueous solution that may play a role to direct the assembly via
templating. Overall, the results show that the combination of specific salts and enzymatic hydrogelation can give rise to complex
self-assembly behaviors that may be exploited to tune hydrogel properties.
derived from the effects on viscosity).42 The other hypothesis
proposes a major role of dispersion forces. These specific ion
effects have later been observed in other areas, such as colloid
INTRODUCTION
■
Peptide-based functional nanomaterials have received extensive
interest as next-generation materials for biomedicine as well as
and surface chemistry, macromolecular systems, such as
energy-related technologies.1−21 A number of strategies have
proteins and polymers, etc.43−51 However, it has only recently
been developed to direct the molecular self-assembly of the
been appreciated in supramolecular chemistry and, more
peptide building blocks, such as changes in the environmental
specifically, for low-molecular-weight (LMW) hydrogels,6,52−54
conditions, including pH, temperature, solvent polarity, ionic
strength, oxidation/reduction state, etc.22−30 An alternative
while a few reports have been published for polymer
hydrogels.55−58
approach is to use a locally applied stimulus, such as light31,32 or
catalytic action of the enzymes.33,34 Enzymatic reactions are
Very recently, we showed that salts can have a pronounced
effect on molecular assembly and resulting material properties
especially useful to direct bottom-up nanofabrication because
of a series of anionic Fmoc-peptide-based gelators, following
they allow for directed self-assembly under otherwise constant
the Hofmeister trend of anions.52 Ions were found to have a
conditions coupled with chemoselective and spatiotemporal
control of nucleation and structure growth.6,7
significant influence on the hydrophobic interactions with
minimal effect on hydrogen-bonding interactions, leading to
Although the “specific ion” effect on protein folding and
more organized supramolecular structures for the kosmotropic
aggregation has been recognized since more than a century ago,
in 1888, by Hofmeister,35 the understanding of this effect still
ions compared to the chaotropic ions. This specific ion effect
provides a powerful parameter that can control the formation of
remains a source of controversy. Hofmeister classified the ions
diverse nanostructures, accessible from a single gelator. Another
according to their relative ability of precipitating hen-egg white
recent example includes a macrocyclic LMW hydrogelator,
protein in aqueous solution, resulting in the sequence shown in
proline-functionalized calix-[4]-arene, which showed ion-
Figure 1a. Two basic hypotheses have been put forward to
account for this phenomenon.36−41 One is attributed to the
bulk effect, which involves the ability of the ions to make and
break hydrogen bonds with water (kosmotropes and chaot-
ropes, characterized by their Jones−Dole viscosity B coefficient,
Received: August 22, 2012
Revised: October 31, 2012
Published: November 1, 2012
© 2012 American Chemical Society
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Figure 1. Biocatalytic self-assembly and gelation in the presence of salts. (a) Order of ions by their B coefficients. The placing of phosphates is
debatable because of the presence of both H2PO4− and HPO42− at near-neutral pH. (b) Subtilisin triggered hydrolysis of the Fmoc-YL-OMe to form
hydrogelator Fmoc-YL.
specific sol−gel transition, but the effect of the salts on the
molecular organization of the hydrogel was not studied.53 Very
recently, Adams et al. demonstrated a cation-specific effect on
an anionic aromatic peptide gelator, where the electrostatic
(cross-linking) interactions between the cation and gelator were
shown to be important.54
Herein, we combine biocatalysis and molecular self-assembly
in the presence of different salts to access diverse supra-
molecular nanostructures. We reveal a dual, cooperative effect
of the salts on the molecular self-assembly of a Fmoc-dipeptide
hydrogelator: (i) differential structuring of the enzyme
molecules in the presence of different salts that affects the
enzyme kinetics and may act as a template for self-assembly, in
addition to the (ii) inherent salt effects on the self-assembly of
the peptide hydrogelator. As might be expected, supramolecular
Figure 2. Time-dependent HPLC of the enzyme reaction with
different salts.
order correlated with the presence of the kosmotropic salts
compared to the chaotropic salts. Interestingly, we observed an
unexpected effect on the handedness of the fibrillar structure
with chiral inversion observed in the final gel-phase material.
further spectroscopic and structural analyses were performed
after 24 h.
Self-assembly of these aromatic peptide amphiphiles is driven
by noncovalent intermolecular interactions (π−π stacking,
hydrogen-bonding interactions, etc.) in addition to the
hydrophobic effect.59−62 Such molecular level interactions can
be followed by a number of spectroscopic methods. Our recent
study on the dynamics of the self-assembly process has shown
that molecular order was induced during enzyme conversion,
driven by the hydrophobic effect and π-stacking interaction
between the fluorenyl groups; it is locked into a β-sheet
structure upon cooling.6 Therefore, we will focus on the effects
of the salts over supramolecular organization of fluorenyl
groups.
Fluorescence emission spectra showed two characteristic
peaks (Figure 3a) corresponding to the monomer and the
highly π-stacked structure. The peak with a maximum at 320
nm corresponds to the monomeric Fmoc-YL emission, while
the relatively broad peak at 420−440 nm is due to the excimer
formation, owing to the π-stacking interactions between the
fluorenyl groups. The intensity of the monomeric emission was
found to be progressively quenched with respect to excimer
emission in the expected order (chaotropes to kosmotropes).
However, a red-shifted monomeric peak (340 nm) was
observed for the Fmoc-YL gels in the presence of nitrate ion,
RESULTS AND DISCUSSION
■
Biocatalytic Self-Assembly. The self-assembled system
used in this study is based on an aromatic peptide amphiphile,
Fmoc-tyrosine-leucine methyl ester (Fmoc-YL-OMe; Figure
1b).52 Molecular self-assembly is induced by the hydrolysis of
the methyl ester by an esterase, subtilisin, which cleaves the
methyl ester to form the peptide derivative, Fmoc-YL, that self-
assembles through a π-stacking interaction between the
fluorenyl groups and finally interlocked into an antiparallel β-
sheet structure, referred to as the π−β structure. The sodium
salts given in Figure 1 were added at a concentration of 100
mM (Figure 1b). In each case, after 1 h of incubation at 55 °C,
followed by cooling to room temperature (20 °C), self-
supporting gels were formed. HPLC time courses revealed that
the rate of biocatalysis was significantly affected by the presence
of the salts and followed the relative ordering of the ions in
Hofmeister series. Such an ion-specific effect on biocatalytic
activity is well-known for a number of enzymes.43−46 In each
case, near-complete conversion was observed within 1 h
(Figure 2), and therefore, final gelator concentrations were
identical in each case. To ensure the complete conversion,
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Figure 3. Spectroscopic evidence of induced supramolecular order in the presence of different salts for biocatalytic and chemical self-assembly. (a)
Emission spectra (excitation at 280 nm) of Fmoc-YL gel obtained with different salt additives. (b) Emission ratio (ratio of intensity of the excimer to
the monomer) for the biocatalytic and chemical self-assembly. (c) CD spectra of supramolecular hydrogel of Fmoc-YL, showing that higher order
chirality is induced following the Hofmeister series. (d) Increase in the ellipticity at 303 nm from chaotropes to kosmotropes for the biocatalytic and
chemical self-assembly.
suggesting a different supramolecular organization. The
emission ratio, which is the relative intensity of the excimer
to the monomer emissions, showed a higher value for the
kosmotropes than chaotropes (Figure 3b), suggesting that
kosmotropes promote the formation of extended π−π
interactions among the fluorenyl moieties and, thus, inducing
more order in the self-assembled state. Importantly, the effects
of salts were found to be significantly enhanced for biocatalytic
self-assembly compared to the chemical self-assembly,52 shown
in Figure 3b.
It is well-known that the formation of supramolecular chiral
structures gives rise to intense CD signals. Intense CD signals
have been shown previously in Fmoc-dipeptide gels, and it
originates principally from the supramolecular organization of
the Fmoc substituent (in addition to contributions from the
amide backbone and tyrosyl side chains).59−62 CD spectra were
recorded for Fmoc-YL gels in the presence of different salts,
which showed a characteristic peak at 303 nm, corresponding
to the fluorenyl absorption.6 Figure 3c showed that all of these
gels gave rise to a negative signal, which indicates the single
handedness for the supramolecular structures. The extent of
induced chirality, as shown in Figure 3d, was significantly
affected by the salts; more intense CD was observed for
kosmotropes compared to chaotropes. Surprisingly, we
observed a chiral inversion in the enzyme-triggered self-
assembly compared to the chemical self-assembly, as reported
earlier.52 We investigated whether these effects could be related
to the kinetic effects of biocatalytic self-assembly and
hydrogelation (Figure 2), as described in the next section.
However, to assess whether the spectroscopic properties were
affected by scattering, we have carried out the turbidity
measurement (see Figure S1 of the Supporting Information),
which showed the absence of significant interference from
scattering.
To gain insight into the nanoscale morphological properties
of the gels formed, atomic force microscopy was performed on
Fmoc-YL gels produced in the presence of the sodium salts of
phosphate (kosmotrope) and thiocyanate (chaotrope) (see
Figure S2 of the Supporting Information). Remarkable
morphological differences were observed. Gels formed in the
presence of phosphate revealed a fibrous morphology, with
dimensions ranging from 95 to 125 nm. In contrast, for
thiocyanate less organized spherical aggregates were found to
be formed of dimensions ranging from 150 to 600 nm. The
formation of spherical aggregates rather than unidirectional
fibers (see Figure S2a of the Supporting Information) for
thiocyanate-containing gels (see Figure S2b of the Supporting
Information) is in agreement with the corresponding reduction
of the emission ratio and CD signal for thiocyanate.
Determination of the gel melting temperature also supported
that the gels were structurally different. Tgel varies from 50 to 58
°C between the extreme chaotropes and kosmotropes (Figure
4a). The mechanical properties of these gels were then probed
by oscillatory rheology. For all gels, the storage modulus (G′)
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Figure 4. Thermal stability, mechanical strength, and spectroscopic behavior of the gels formed catalytically (black), by a heating−cooling cycle
(red), and chemically (blue). (a) Melting temperature (Tgel) of Fmoc-YL gels formed in the presence of different salts. (b) Mechanical strength of
Fmoc-YL gels with different salts as measured by oscillatory rheology. (c) Emission ratio (ratio of intensity of the excimer to the monomer) with
different salts. (d) Increase in the ellipticity at 303 nm from chaotropes to kosmotropes.
was found to be an order of magnitude higher than the loss
modulus (G″). G′ varies from 0.3 to 3 KPa throughout the
series (Figure 4b).
To investigate the possible templating effect of the enzyme,
owing to their structuring/clustering in aqueous solution, as
shown previously,63,64 SLS was employed in the presence of
different ions. The background-corrected SLS intensities are
shown in Figure 5. The subtilisin molecules cluster to form
Thermodynamics versus Kinetics in Biocatalytic
Assembly. After melting of the gels at 90 °C and cooling
back to room temperature, all of the gels show a more
pronounced salt-induced order in their self-assembled state, as
reflected in their mechanical strength and induced supra-
molecular chirality (Figure 4 and Figure S3 of the Supporting
Information). Although we did not find much difference in the
thermal stability of the gels with different salts after the heat/
cool cycle (Figure 4a), a pronounced effect was observed in the
mechanical strength of the gels (Figure 4b), suggesting different
self-assembled network structures. We also did not observe
substantial differences in the emission ratio after heat/cool
compared to the catalytic induction. An enhancement in the π-
stacking interaction was observed, which reflected the enhanced
salt-induced effect on the supramolecular chirality of the gels. A
much higher emission ratio was again observed for phosphate
ion, which is unexpected and will require further investigation.
This observation indicates that, under catalytic control,
kinetically trapped structures were formed, which access a
more thermodynamically favored state upon heating/cooling.
Salts, therefore, induced dual control of the biocatalytic self-
assembly process as they influenced enzyme performance
(affecting kinetics) as well as molecular level interactions
between the gelator molecules and water molecules.
Figure 5. SLS intensities of subtilisin in the presence of different salts.
large aggregates at 55 °C, having a radius of gyration (Rg) of
approximately 253 nm in the presence of kosmotropic salt,
sodium citrate. The power law analysis used to characterize the
fractal dimension exponent (df) of the clusters indicated the
scattering pattern scales with an exponent of 1.5 in the Q region
above 0.005 nm−1, which indicates that the enzyme clusters
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reversible gel−sol transition temperature (Tgel) was determined using
a vial inversion method at varying temperatures.
form fairly open mass fractal structures in the presence of
citrate ions (see Figure S4 of the Supporting Information). The
power law exponent in the Q region below 0.005 nm−1 is
around 1, suggesting that the subtilisin fractal clusters extend in
one dimension to form structures similar to short rods. The
scattering pattern of subtilisin in the presence of sodium
phosphate resulted in a power law exponent of 1.6 in the whole
range of scattering vector Q. It indicates that the fairly open
mass fractal structures are present at all of the length scales. In
contrast to the kosmotropic ions, the chaotropic thiocyante ion
has different effects on the clustering of subtilisin molecules.
The power law exponent changes from 1.6 (in the case of
kosmotropic ions) to 2.5, which indicates the presence of dense
mass fractal structures of enzymes, which are presumably rigid
and less dynamic and may have buried active sites of enzyme in
the presence of chaotropic thiocyanate ions (see Figure S4 of
the Supporting Information).
These results suggest that the structuring of the subtilisin
molecules plays an important role in the self-assembly of the
Fmoc-YL hydrogelator. The kosmotropic ions favor the open
and dynamic fractal networks of the enzymes, but the
chaotropic ion generates a dense mass fractal structure. It is
highly likely that these fractal structures also play a role as a
template for self-assembly of the hydrogelator along with its
enzymatic action.
Fluorescence Spectroscopy. Fluorescence emission spectra were
measured on a Jasco FP-6500 spectrofluorometer with light measured
orthogonally to the excitation light, at a scanning speed of 100 nm
min−1. The excitation wavelength was 280 nm, and emission data were
recorded in the range between 300 and 600 nm. The spectra were
measured with a bandwidth of 5 nm with a medium response and a 1
nm data pitch. Quartz cells with a path length of 10 mm were used for
the study.
Circular Dichroism (CD). Spectra were measured on a Jasco J600
spectropolarimeter with 1 s integrations with a step size of 1 nm and a
single acquisition with a slit width of 1 nm because of the dynamic
nature of the system. Quartz cells of 0.2 mm path length were used for
the measurement.
High-Performance Liquid Chromatography (HPLC). A Dionex
P680 HPLC pump was used to quantify conversions of the enzymatic
reaction. A 50 μL sample was injected onto a Macherey-Nagel C18
column of 250 mm length with an internal diameter of 4.6 mm and 5
mm fused silica particles at a flow rate of 1 mL min−1 [eluting solvent
system: linear gradient of 20% (v/v) acetonitrile in water for 4 min
and gradually rising to 80% (v/v) acetonitrile in water at 35 min; this
concentration was kept constant until 40 min when the gradient was
decreased to 20% (v/v) acetonitrile in water at 42 min]. The sample
preparation involved mixing 50 μL of gel with acetonitrile−water (950
μL, 50:50 mixture) containing 0.1% trifluoroacetic acid. The purity of
each identified peak was determined by the ultraviolet (UV) detection
at 280 nm.
Oscillatory Rheology. To verify the mechanical properties of the
resulting hydrogels, dynamic frequency sweep experiments were
carried out on a strain-controlled rheometer (Kinexus Pro rheometer)
using parallel-plate geometry (20 mm diameter). The experiments
were performed at 25 °C, and this temperature was controlled
throughout the experiment using an integrated electrical heater.
Additional precautions were taken to minimize solvent evaporation
and to keep the sample hydrated: a solvent trap was used, and the
internal atmosphere was kept saturated. To ensure that the
measurements were made in the linear viscoelastic regime, an
amplitude sweep was performed and the results showed no variation
in elastic modulus (G′) and viscous modulus (G″) up to a strain of 1%.
The dynamic modulus of the hydrogel was measured as a frequency
function, where the frequency sweeps were carried out between 0.1
and 100 Hz. The gels were made in small fractions in wide-mouth vials
from which they were transferred with a spatula for rheological
measurements. The measurements were repeated 3 times to ensure
reproducibility, with the average data shown.
CONCLUSION
■
We demonstrated that salts have a dramatic effect on the
molecular self-assembly of the aromatic dipeptide amphiphiles
in aqueous media. Salts affect the structuring of the enzyme
network, which, in turn, influences the enzyme kinectics and
the corresponding nucleation and growth of the nanostructures.
This is manifested in the differential hydrophobic interactions,
resulting in differential order and chirality as well as variable
mechanical properties in the resulting gel-phase materials. The
order of efficiency of the ions to promote such interactions
followed the Hofmeister trend. Such a specific ion effect can be
attributed to the complex interplay of both the electrostatic and
hydrophobic interactions. Although the exact mechanism
behind this effect is not fully understood, it can be rationalized
by considering electrostatic screening of the charges, ion
binding, and its implication to hydrophobic interactions, as
discussed in our recent work.52 The present study demonstrates
that highly complex self-assembly behavior occurs when
combining the effects of salts, protein templating, and catalysis.
Insight into these behaviors is useful in controlling and
directing peptide self-assembly toward desired nanomaterials.
Static Light Scattering (SLS). SLS measurements were carried
out using the 3DDLS instrument (LS Instruments, Fribourg,
Switzerland) using vertically polarized He−Ne laser light (25 mW
with a wavelength of 632.8 nm) with an avalanche photodiode
detector at angles between 15° and 135° at 55 °C. The background
scattering intensities (buffers with added salts) were subtracted from
the scattering intensities of the enzyme solutions for further analysis.
The scattering intensity patterns from static light and X-ray scattering
experiments can be described as I(Q) ∼ KP(Q)S(Q), where K is an
instrument- and sample-dependent constant, P(Q) is the form factor
that depends upon the size and shape of the primary particles, and
S(Q) is the structure factor giving information about the spatial
arrangement of the primary particles at length scales larger than that of
the primary particles (radius Rp). In the limit of QRg < 1, the mean
radius of gyration Rg of randomly distributed (e.g., freely diffusing)
primary particles or clusters can be determined from the measured
scattered intensity I(Q) using the Guinier analysis. In the limit of 1/Rg
≪ Q ≪ 1/Rp, where Rg is the mean radius of gyration of a sufficiently
large cluster composed of primary particles with radius Rp, the
structure factor for fractal clusters with fractal dimension df scales with
EXPERIMENTAL SECTION
■
Catalytic Self-Assembly. Fmoc-YL-OMe (10 mmol/kg) was
dispersed in a 1 mL volume of 100 mM sodium phosphate buffer (pH
8) in the presence of different sodium salts (100 mM) and 30 μL of
subtilisin from Sigma-Aldrich (catalogue number P4860; LOT
056K1213) within a 10 mm sample vial. The mixture was vortexed
(30 s) and sonicated on ice for 20 min to ensure that a homogeneous
mixture was obtained, while the low temperature assures that no
enzymatic conversion occurs up to this point. This was followed by
heating in an oil bath at 55 °C for 60 min to allow for the enzymatic
conversion to occur. The self-assembling system was then allowed to
cool to room temperature. The gel samples were then left overnight
before experimental measurements were performed. Gelation was
considered to have occurred when a homogeneous “solid-like” material
was obtained that exhibited no gravitational flow. The thermally
Q through a power law relation as I(Q) ∼ S(Q) ∼ Q−d . The radius of
f
gyration can be calculated by plotting Q2 and ln I(Q), and then linear
regression will give the slope, from where we can calculate Rg. The
power law behavior and fractal dimension are calculated by plotting
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ASSOCIATED CONTENT
* Supporting Information
■
S
Turbidity data, AFM images of the gels with different salts.
Fluorescence emission spectra of Fmoc-YL gels after the heat/
cool cycle and open and rigid mass fractal structure of the
enzyme in the presence of kosmotropic and chaotropic salts.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: rein.ulijn@strath.ac.uk (R.V.U.); sangita.roy@strath.
ac.uk (S.R.).
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the EPSRC, HFSP, ERC (Starting Grant EMERgE),
and Leverhulme Trust (Leadership Award) (U.K.) for funding.
The material is based on research sponsored by the Air Force
Laboratory, under Agreement FA9550-11-1-0263.
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Extensive neurite outgrowth and active synapse formation on self-
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E. W. Probing the solvent-assisted nucleation pathway in chemical self-
assembly. Science 2006, 313, 80.
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