Self-assembly of a catalytic multivalent peptide-nanoparticle complex

Article abstract of DOI:10.1021/ja302754h

Catalytically active peptide-nanoparticle complexes were obtained by assembling small peptide sequences on the surface of cationic self-assembled monolayers on gold nanoparticles. When bound to the surface, the peptides accelerate the transesterification

Full text of DOI:10.1021/ja302754h

Communication
pubs.acs.org/JACS
Self-Assembly of a Catalytic Multivalent Peptide−Nanoparticle
Complex
Davide Zaramella, Paolo Scrimin,* and Leonard J. Prins*
Department of Chemical Sciences, University of Padova, Via Marzolo 1, 35131 Padova, Italy
S
* Supporting Information
amply demonstrated the attractiveness of cationic Au MPCs as
ABSTRACT: Catalytically active peptide−nanoparticle
complexes were obtained by assembling small peptide
sequences on the surface of cationic self-assembled
monolayers on gold nanoparticles. When bound to the
surface, the peptides accelerate the transesterification of
the p-nitrophenyl ester of N-carboxybenzylphenylalanine
by more than 2 orders of magnitude. The gold
nanoparticle serves as a multivalent scaffold for bringing
the catalyst and substrate into close proximity but also
creates a local microenvironment that further enhances the
catalysis. The supramolecular nature of the ensemble
permits the catalytic activity of the system to be modulated
in situ.
a construction element for the development of innovative
biosensors.11−13 Here we now show that the self-assembly of
histidine-containing peptides H1−H3 on the surface of Au
MPC 1 triggers their esterolytic activity (Figure 1), resulting in
he catalytic efficiency, mechanistic pathways, and
Tstructural complexity displayed by enzymes make them
a tremendous source of inspiration for chemists involved in
catalyst development.1,2 Nature has evolved enzymes as large
multi-kilodalton complex structures in which even units that are
remote from the actual active site may profoundly affect the
activity of the enzyme.3 The much lower complexity of artificial
enzyme mimics may be an important reason for their typical
modest performances with respect to enzymes. This awareness
has led to an interest in catalysts based on multivalent scaffolds,
such as dendrimers,4 micelles,5 and nanoparticles,6 with the
idea of increasing the structural complexity of the synthetic
system. A key challenge is straightforward access to synthetic
catalysts that can match up to the size and complexity of
enzymes. The necessity for multistep synthesis can be
overcome by relying on self-assembly for the formation of
the multivalent structure. In particular, the self-assembly of
catalytic monolayers on the surface of gold nanoparticles (Au
NPs) to give gold monolayer-protected clusters (Au MPCs) is
emerging as an attractive strategy.7,8 Nonetheless, although they
rely on self-assembly, the composition of self-assembled
monolayers (SAMs) on Au NPs is typically still of rather low
complexity.9 This mainly originates from the use of synthetic
protocols for mixed SAM formation (e.g., place exchange),
which do not give full control over the final composition,
require purification of each single NP system, and suffer from
issues related to the characterization of mixed SAMs both in
terms of composition and morphology. For that reason, we
recently started to study the formation of heterofunctionalized
multivalent structures relying on the self-assembly of small
peptides on the surface of Au MPCs.10 Those studies extended
the seminal contributions by Rotello and co-workers, which
Figure 1. Self-assembly of peptides H0−H3 on the surface of Au MPC
1, resulting in the formation of Hx/Au MPC 1 complexes that (for x =
1−3) can catalyze the transesterification of the substrate Cbz-Phe-
ONP.
nanostructures that can induce a more than 100-fold rate
acceleration. Self-assembly is a prerequisite for catalysis, as the
peptides are not catalytically active at all in the absence of the
Au MPCs. Importantly, the multivalent surface plays a crucial
role in tuning the catalytic activity. The surface not only brings
the substrate and catalyst in close proximity but also generates a
microenvironment with an enhanced local pH that further
activates the catalytic peptide.
We previously showed that the (covalent) insertion of
imidazole residues in a SAM generates nanosystems that act as
transesterification catalysts.14 It occurred to us that cationic Au
MPCs would be an excellent multivalent platform for bringing
such catalytic units together through self-assembly. Peptides
H1−H3 were designed taking into consideration three issues:
(a) the presence of three aspartic acid residues for binding to
Au MPCs 1, (b) the presence of a fluorescent tryptophan
residue to verify the binding, and (c) the presence of one or
more histidine residues for catalysis. Peptide H0 lacking
histidine units was added as an inert reference compound. Au
MPCs 1 (1.8 0.3 nm diameter) containing trimethylammo-
nium headgroups were prepared and characterized as described
Received: March 21, 2012
Published: May 4, 2012
© 2012 American Chemical Society
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previously.10,15 Binding of peptides to Au MPCs 1 was studied
using fluorescence titrations, relying on the ability of the Au
16
NPs to quench the fluorescence of bound fluorophores.
Thus, the fluorescence intensity of tryptophan emission was
measured as a function of the amount of peptide H0−H
3
added to a solution of Au MPC 1 ([headgroup] = 60 μM) in
water buffered at pH 7.0. The obtained profiles (Figure 2) are
Figure 3. (a) Changes in the absorbance at 400 nm upon the addition
□
of Cbz-Phe-ONP (10 μM) to solutions of Au MPC 1 ( ), H0/Au
■
■
■
MPC 1 (black ), H1/Au MPC 1 (red ), H2/Au MPC 1 (blue ),
■
and H3/Au MPC 1 (green ). Included also are data for the
background hydrolysis of Cbz-Phe-ONP at the same concentration
○
( ). Solid lines represent the best fits to a kinetic model (see the SI).
Conditions: [Au MPC 1 headgroup] = 60 μM; [H0] = 11 μM, [H1] =
11 μM, [H2] = 8.5 μM, [H3] = 8.5 μM; [HEPES] = 10 mM; H2O/
CH3CN = 90:10; pH 7.0; T = 37 °C. (b) Observed rate constants
(kobs), rate accelerations relative to background (kobs/kuncat), and rate
constants corrected for the surface saturation concentration of peptide
H0−H3 (k2). (c) Plot of the second-order rate constant k2 as a function
of the number of histidines present in peptides H0−H3.
Figure 2. Tryptophan fluorescence intensity at 360 nm as a function of
the concentration of peptides H0− H3 in the presence of Au MPC 1
([headgroup] = 60 μM). Conditions: pH 7.0; [HEPES] = 10 mM;
H2O/CH3CN = 90:10; T = 37 °C). The solid lines represent best fits
to a binding model used to determine the surface saturation
concentration (concsat) values shown in the inset.11 The slightly
different slopes in the linear parts of the curves result from the
different intrinsic fluorescence properties of the tryptophan residues in
H0−H3 (see the SI).
straight line (Figure 3c). This shows that the histidine residues
are indeed at the origin of the catalytic effect. Furthermore, the
linear correlation indicates that the catalysis does not originate
from the cooperative action between two histidine residues in
the same probe.
Regrettably, the low solubility of the substrate did not permit
us to perform catalytic experiments using large excesses of
substrate. To show that the catalytic system H1/Au MPC 1
indeed works under turnover conditions, this problem was
solved by sequentially adding batches of Cbz-Phe-ONP
substrate at the saturation concentration of 10 μM. The
amount of PNP released was measured as a function of time
after each addition, yielding the profile depicted in Figure 4.
Since H1 was present at a concentration of 5 μM (see below),
the total release of 40 μM PNP shows that H1/Au MPC 1
indeed operates under turnover conditions. The slight decrease
in reactivity observed after each addition presumably originates
from competitive binding of the reaction products. The
indicative of strong complex formation between the peptides
and Au MPC 1. From the binding isotherms, a surface
saturation concentration (concsat) was determined using a
procedure reported previously.10 The surface saturation
concentration defines the maximum loading of the peptide
on the Au MPC surface (Figure 2 inset).17
Subsequently, we tested the catalytic activity of the obtained
systems in the transesterification of the p-nitrophenyl ester of
N-carboxybenzylphenylalanine (Cbz-Phe-ONP).18 Kinetic ex-
periments were performed by adding the substrate (10 μM) to
a solution of Au MPC 1 and the peptides H1−H3 at their
respective surface saturation concentrations in 9:1 H2O/
CH3CN buffered at pH 7.0 at 37 °C. The presence of 10%
CH3CN was required for solubilization of the substrate. The
kinetics of hydrolysis were followed by measuring the increase
in absorbance at 400 nm originating from the release of the p-
nitrophenolate anion (PNP). We were excited to observe that
all of the complexes H1−3/Au MPC 1 gave rise to rate
accelerations of at least 2 orders of magnitude for the cleavage
of Cbz-Phe-ONP relative to the background reaction (Figure
3a,b). The observed change in absorbance of 0.1 units at 400
nm corresponded to that expected for the complete hydrolysis
of 10 μM substrate. Importantly, the addition of any one of the
peptides H1−H3 at the same concentrations but in the absence
of Au MPC 1 did not result in any rate acceleration over the
background (see the SI). The obtained rate constants were
corrected for the surface saturation concentration in order to
compare the contributions to catalysis by the various peptides.
A plot of the obtained second-order rate constants as a function
of the number of histidines present in the catalyst gave a
Figure 4. Amounts of PNP released upon four consecutive additions
of Cbz-Phe-ONP substrate (10 μM) to a solution of H1/Au MPC 1 as
a function of time. Conditions: [Au MPC 1 headgroup] = 60 μM;
[H1] = 5 μM; [HEPES] = 10 mM; H2O/CH3CN = 90:10; pH 7.0; T
= 37 °C.
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Communication
experiments also show that there is no accumulation of
intermediates during the reaction.
To understand the origin of the catalytic effect, a series of
experiments were performed. Interestingly, it was observed that
the simple addition of Au MPC 1 to Cbz-Phe-ONP also
resulted in a 20-fold enhancement in the hydrolysis rate (□ in
Figure 3a), which points at a catalytic role of the cationic
headgroups. The addition of tetramethylammonium chloride at
the same concentration had no effect at all (see the SI). Similar
effects have also been observed in cationic micelles and ascribed
to an increase in the local pH where the substrate binds.19−21
Experimental evidence that the pH at the monolayer surface is
indeed higher than the bulk pH was obtained by adding the pH
indicator bromothymol blue (30 μM) to a solution of Au MPC
1 under the exact conditions of the catalytic experiments (i.e.,
pH 7.0). Bromothymol blue (pKa = 7.1) has two negative
charges under basic conditions. This, combined with the
presence of a large hydrophobic aromatic portion, was expected
to generate a significant binding affinity for Au MPC 1.
Comparison of the absorption spectra in the presence and
absence of Au MPC 1 indeed showed a significant increase of
0.29 units in the absorption maximum at 620 nm,
corresponding to an apparent local pH of 7.7 (see the SI).
Importantly, the catalytic effect of Au MPC 1 is to a large
extent suppressed upon addition of the reference peptide H0 at
the surface saturation concentration of 11.0 μM (■ in Figure
3a). Thus, the reference compound H0 neutralizes the effect of
the trimethylammonium groups on the pH. These observations
were confirmed by an experiment in which peptides H0 and H1
were added 300 s after mixing Au MPC 1 and the substrate
(Figure 5a). Relative to the control (no addition), the resulting
profiles clearly indicate the accelerating effect of H1 and the
inhibitory effect of H0 on the catalysis. In addition, this
experiment also illustrates an important aspect of supra-
molecular catalysts, which is the ability to modulate the activity
in situ as a function of the type of components added.
Figure 5. (a) Changes in the absorbance at 400 nm as functions of
■
time for the experiments in which either H0 (11 μM, black ) or H1
■
(11 μM, red ) was added 300 s after mixing of Au MPC 1 (60 μM,
□
) and Cbz-Phe-ONP (10 μM). Conditions: [HEPES] = 10 mM;
H2O/CH3CN = 90:10; pH 7.0; T = 37 °C. (b) Observed rate
constants (kobs) as functions of the concentrations of peptide H1 (red
■
■
and yellow ) or H0 (black ) at a constant concentration of Au
MPC 1 (60 μM) and Cbz-Phe-ONP (10 μM). The yellow symbols
were obtained in the experiment performed with [H0] + [H1] held
constant at 11 μM. Conditions: [HEPES] = 10 mM; H2O/CH3CN =
90:10; pH 7.0; T = 37 °C.
The high local pH indicates that cooperativity between
imidazoles localized on different probes, as is frequently
observed in multivalent imidazole-containing catalysts,14,22,23
is unlikely to be the source of the observed catalytic activity, as
this would require both protonated and unprotonated
imidazoles. This was indeed confirmed by measuring the rate
of hydrolysis of Cbz-Phe-ONP as a function of the amount of
H1 added to Au MPC 1 over the range from 0 to 11 μM. In the
case of positive cooperativity, an exponental profile would be
expected, corresponding to the exponential formation of
catalytic sites composed of two imidazoles.24,25 Rather, the
obtained profile was exactly the opposite of that, showing a
strong increase in kobs at low H1 concentrations (up to 4 μM)
and constant values afterward (red ■ in Figure 5b). Evidently,
the highest catalytic activity per histidine residue is obtained not
at maximum surface saturation of Au MPC 1 by H1 but rather
in a situation in which the peptide is isolated on the surface.
Thus, the obtained profile indicates an active contribution of
the cationic SAM to the catalysis. Indeed, the results are fully
consistent with the hypothesis that the presence of a higher
local pH on the SAM affects the concentration of deprotonated
nucleophilic imidazole. As the peptide covers the surface, its
carboxylates replace the OH− counterions of the ammonium
headgroups, thus decreasing the local pH.21 This in turn
increases the concentration of protonated imidazole. Evidence
for this hypothesis was obtained by an analogous experiment in
which the contribution of the monolayer was suppressed by
performing the measurements in the presence of a compensat-
ing amount of H0 (i.e., [H0] + [H1] held constant at 11 μM;
yellow ■ in Figure 5b). In this case, the increase in kobs as a
function of [H1] was linear over the full interval explored, but
the slope was smaller than that observed in the first part of the
previous plot, accounting for a lower deprotonated imidazole
concentration in this case. Also, the observation that the
reaction rate decreased when the pH was lowered (see the SI)
is consistent with this explanation, since a cooperative
mechanism would have led to an increase in the rate resulting
from the presence of both protonated and nonprotonated
imidazoles. Similar changes in the reactivity of imidazoles
because of a different local chemical environment have also
been observed in other multivalent systems.26,27
The above experiment indicates that the increase in local pH
due to the cationic monolayer can explain the higher activity of
H1 at lower surface loadings but not at the surface saturation
concentration, where this effect is largely suppressed. A final
piece of information came from measurements of the initial
reaction rate as a function of Cbz-Phe-ONP concentration
(Figure 6). Although the solubility of the substrate permitted
only a relatively small range (1−10 μM) to be studied, the
nonlinearity of the plot of the initial rate versus substrate
concentration gave clear evidence of substrate binding to Au
MPC 1. We hypothesize that this binding is driven by
hydrophobic interactions between the highly apolar substrate
and the hydrophobic part of the monolayer. Interactions of this
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Journal of the American Chemical Society
Communication
(StG-239898), COST (CM0703 and CM0905), and MIUR is
acknowledged.
REFERENCES
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Figure 6. Initial rate as a function of the concentration of Cbz-Phe-
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type between small molecules and SAMs have also been
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of Au MPC 1 at low micromolar concentrations results in an
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In conclusion, we have developed a supramolecular catalytic
system formed through the self-assembly of small peptides on
the surface of Au MPCs. Self-assembly is a prerequisite for the
catalysis, since the catalytic peptides do not display any activity
in the absence of Au MPCs. Assembly on the surface of Au
MPC 1 results in a rate acceleration of at least 2 orders of
magnitude in a transesterification reaction. The multivalent
surface is essential for bringing the substrate and catalyst into
close proximity but, importantly, also generates a local chemical
environment that enhances the reactivity of the catalytic unit. In
this way, the system mimics some of the key features of
enzymes. The system presented here allows for a series of
exciting possibilities, such as the possibility of fine-tuning the
catalytic properties simply by altering the catalytic peptide
sequence or the possibility of turning the catalytic activity on or
off in situ as a result of the addition of inhibitors/activators. In
our opinion, this approach has the potential to be exploited for
the development of synthetic systems able to match up to the
complexity displayed by enzymes simply by combining various
small fragments.
(16) Dulkeith, E.; Morteani, A. C.; Niedereichholz, T.; Klar, T. A.;
Feldmann, J.; Levi, S. A.; van Veggel, F.; Reinhoudt, D. N.; Moller, M.;
Gittins, D. I. Phys. Rev. Lett. 2002, 89, No. 203002.
(17) The curvature of the binding isotherms measured for H2 and H3
indicated a slightly lower affinity of these peptides for Au MPC 1
attributed to the presence of the (protonated) imidazoles. It was
calculated that upon the addition of 8.5 μM H2 and H3, ∼15% of these
peptides were not bound. For H0 and H1 (at 11 μM) these values were
5 and 8%, respectively.
(18) All studies were performed on enantiomerically pure D-Cbz-
Phe-ONP. Kinetics with the L enantiomer revealed no significant
enantioselectivity.
(19) Tonellato, U. J. Chem. Soc., Perkin Trans. 2 1977, 821.
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(24) Zaupa, G.; Mora, C.; Bonomi, R.; Prins, L. J.; Scrimin, P.
Chem.Eur. J. 2011, 17, 4879.
ASSOCIATED CONTENT
* Supporting Information
Synthesis and characterization of H0−H3, procedures for the
kinetic experiments, background rates and control experiments,
titration with bromothymol blue, and reaction rate as a function
of pH. This material is available free of charge via the Internet
at http://pubs.acs.org.
(25) Zaupa, G.; Scrimin, P.; Prins, L. J. J. Am. Chem. Soc. 2008, 130,
5699.
■
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(26) Pengo, P.; Polizzi, S.; Pasquato, L.; Scrimin, P. J. Am. Chem. Soc.
2005, 127, 1616.
(27) Javor, S.; Delort, E.; Darbre, T.; Reymond, J. L. J. Am. Chem. Soc.
2007, 129, 13238.
(28) Guarino, G.; Rastrelli, F.; Mancin, F. Chem. Commun. 2012, 48,
1523.
AUTHOR INFORMATION
Corresponding Author
paolo.scrimin@unipd.it; leonard.prins@unipd.it
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Alessandro Cazzolaro is gratefully acknowledged for the
synthesis of Au MPC 1. Financial support from the ERC
■
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dx.doi.org/10.1021/ja302754h | J. Am. Chem. Soc. 2012, 134, 8396−8399