Tunable inhibition and denaturation of α-chymotrypsin with amino acid-functionalized gold nanoparticles

Article abstract of DOI:10.1021/ja0512881

Water-soluble gold nanoparticles bearing diverse L-amino acid terminals have been fabricated to probe the effect of receptor surface on protein surface binding. The interaction of these nanoparticles with α-chymotrypsin (ChT) was investigated by activity

Full text of DOI:10.1021/ja0512881

Published on Web 08/23/2005
Tunable Inhibition and Denaturation of r-Chymotrypsin with
Amino Acid-Functionalized Gold Nanoparticles
Chang-Cheng You, Mrinmoy De, Gang Han, and Vincent M. Rotello*
Contribution from the Department of Chemistry, UniVersity of Massachusetts, 710 North
Pleasant Street, Amherst, Massachusetts 01003
Received March 1, 2005; E-mail: rotello@chem.umass.edu
Abstract: Water-soluble gold nanoparticles bearing diverse L-amino acid terminals have been fabricated
to probe the effect of receptor surface on protein surface binding. The interaction of these nanoparticles
with R-chymotrypsin (ChT) was investigated by activity assay, gel electrophoresis, zeta-potential, circular
dichroism, and fluorescence spectroscopy. The results show that both electrostatic and hydrophobic
interactions between the hydrophobic patches of receptors and the protein contribute to the stability of the
complex. The microscopic binding constants for these receptor-protein systems are 106-107 M-1, with
the capacity of the nanoparticle receptors to bind proteins determined by both their surface area and their
surface charge density. Furthermore, it is found that the hydrophilic side chains destabilize the ChT structure
through either competitive hydrogen bonding or breakage of salt bridges, whereas denaturation was much
slower with hydrophobic amino acid side chains. Significantly, correlation between the hydrophobicity index
of amino acid side chains and the binding affinity and denaturation rates was observed.
Introduction
addressed to attain specific protein surface recognition. A few
“small molecule” systems, such as the receptors on calixarene
Protein surface recognition is an important issue in biomedical
and biomaterial research.1 Surface recognition provides a method
complementary to active site inhibition for the control of
enzymatic processes.2 More significantly, protein surface rec-
ognition provides a powerful tool for the regulation of protein-
protein interactions central to a number of cellular processes,
such as cellular signal transduction, DNA transcription, and
protein antigen/antibody recognition.3,4 Protein surface recogni-
tion is an important issue in biosensing, in clinical diagnostics,
and in pharmacology. For example, interruption of protein-
protein interactions has been used by Chmielewski to inhibit
the dimerization and hence activity of HIV-1 protease5 and
Burgess to disrupt NGF-TrkA binding.6
and porphyrin scaffolds,9 cyclodextrin dimers,10 and transition
metal complexes targeted against surface exposed histidines,11
have been recently fabricated to address the challenge for protein
surface recognition and have shown the ability to modulate the
structure and function of biomolecules.
The use of scaffolds with large surface is advantageous,
however, as such receptors could offer larger area of contact
for efficient molecular recognition. In this regard, monolayer-
protected clusters (MPCs) and mixed monolayer-protected
clusters (MMPCs) are promising candidates that provide many
desirable structural attributes for the creation of biomacromo-
lecular receptors. First, a variety of metal and semiconductor
core materials that feature useful fluorescence and magnetic
properties can be used to construct the nanoparticle scaffolds.12
Second, the core sizes of nanoparticles are tunable from 2 to
The required large receptor-protein contact areas7 (usually
>6 nm2) and the inherent complexity of protein surfaces, such
as multiple electrostatic, hydrophobic and hydrogen-bonding
interactions, and complicated topological features,8 must be
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A R T I C L E S
You et al.
>10 nm, which is comparable to that of proteins and other
biomacromolecules, with concomitant large surface areas for
efficient protein binding.13 Third, the surface properties of
nanoparticles can be conveniently tailored by appropriate
selection of ligand functionality, providing versatility in the
creation of surface-specific receptors.14 Indeed, MPCs and
MMPCs have shown interesting application in the multivalent
binding of biomacromolecular targets.15,16
R-Chymotrypsin (ChT), a serine protease of 25 kDa, provides
an excellent system for studying the protein surface recognition
as it is an extensively characterized protein with well-defined
geometry, surface “hot spot”, and enzymatic activity. Our
previous investigations have demonstrated that simple carboxylic
acid-terminated nanoparticles (with either Au or CdSe core)
could target ChT through complementary electrostatic interac-
tions, resulting in the inhibition of ChT activity.17 However, to
allow more pragmatic uses of nanoparticles for regulation of
protein-protein interaction, a prerequisite is the modification
of the nanoparticle surface to better mimic protein surface
functionality. This complexity would provide control over
protein specificity and protein stability, giving insight into issues
important in modulating protein-protein interactions such as
complementary electrostatic interaction, hydrophobic interaction,
and steric effect. Furthermore, through these studies we would
be able to address other crucial issues, such as the factors
contributing to stabilization/denaturation of MMPC-bound
proteins.
Amino acids provide an attractive means for generating
structural diversity.18 In the current study, we have fabricated a
series of L-amino acids-functionalized gold nanoparticles and
examined their interaction with ChT (Figure 1). The amino acid-
decorated surfaces represent the simplest mimics of protein
surfaces. Their interaction with proteins thus exhibits much more
resemblance with the naturally occurring protein-protein
systems. In this study, we have found that the interactions
between ChT and these MMPCs depend on the surface
Figure 1. (a) Amino acid-decorated cluster surface that consists of
carboxylic acid recognition elements as well as extra functions for
perturbation. (b) The relative sizes of amino acid-functionalized gold
nanoparticles and ChT. The surface of ChT is patterned with the electrostatic
surface potential, showing basic and acidic domains on protein surface.
composition of nanoparticles. Significantly, the binding affinity
as well as the stability of the protein is regulated by the charge
and the hydrophobicity of amino acid side chains in the
nanoparticles. Therefore, it is possible to control the association/
dissociation and stabilization/denaturation of the protein at the
MMPC interfaces through variation of the extra functionality
in the vicinity of carboxylic acid recognition elements.
Results and Discussion
Fabrication of Amino Acid-Functionalized Gold Nano-
particles. In the past decade, ω-functionalized n-alkanethiols
have been extensively used as building blocks in self-assembled
monolayers on gold.19 While a number of oligopeptide-
functionalized thioligands have been designed and synthesized,20
amino acid-terminated thiol ligands have rarely been reported,21
although the latter provides more fundamental information on
the monolayer construction. For example, variation in amino
acid side chain structure will allow us to probe the effect of
hydrophobic groups (e.g., Leu, Val), additional anionic func-
tionality (e.g., Asp, Glu), cationic groups (e.g., Arg), and
hydrogen-bonding functionality (e.g., Asn, Gln).
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C.-J.; Harris, J. E.; Vachet, R. W.; Clark, M. R.; Londono, J. D.; Green, S.
J.; Stokes, J. J.; Wignall, G. D.; Glish, G. L.; Porter, M. D.; Evans, N. D.;
Murray, R. W. Langmuir 1998, 14, 17-30.
(14) (a) Li, X.-M.; Huskens, J.; Reinhoudt, D. N. J. Mater. Chem. 2004, 14,
2954-2971. (b) Templeton, A. C.; Wuelfing, M. P.; Murray, R. W. Acc.
Chem. Res. 2000, 33, 27-36.
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104, 293-346. (c) Pasquato, L.; Pengo, P.; Scrimin, P. J. Mater. Chem.
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2004, 43, 6042-6108. (e) Shenhar R.; Rotello, V. M. Acc. Chem. Res.
2003, 36, 549-561. (f) Sastry, M.; Rao, M.; Ganesh, K. N. Acc. Chem.
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5932-5933. (b) Verma, A.; Nakade, H.; Simard, J. M.; Rotello, V. M. J.
Am. Chem. Soc. 2004, 126, 10806-10807. (c) Zheng, M.; Huang, X. J.
Am. Chem. Soc. 2004, 126, 12047-12054. (d) Raschke, G.; Kowarik, S.;
Franzl, T.; So¨nnichsen, C.; Klar, T. A.; Feldmann, J.; Nichtl, A.; Ku¨rzinger,
K. Nano Lett. 2003, 3, 935-938. (e) Wilson, R. Chem. Commun. 2003,
108-109. (f) Wang, G.; Zhang, J.; Murray, R. W. Anal. Chem. 2002, 74,
4320-4327. (g) Cao, Y. C.; Jin, R.; Mirkin, C. A. Science 2002, 297,
1536-1540. (h) McIntosh, C. M.; Esposito, E. A.; Boal, A. K.; Simard,
J. M.; Martin, C. T.; Rotello, V. M. J. Am. Chem. Soc. 2001, 123,
7626-7629. (i) Nath, N.; Chilkoti, A. J. Am. Chem. Soc. 2001, 123, 8197-
8202.
We have shown that carboxylate-functionalized nanoparticle
clusters bind ChT and inhibit its activity through complementary
electrostatic interaction.17 In further studies, it has been revealed
that tetra(ethylene glycol) tethers in the nanoparticles minimize
(19) Witt, D.; Klajn, R.; Barski, P.; Grzybowski, B. A. Curr. Org. Chem. 2004,
8, 1763-1797.
(20) (a) Pengo, P.; Polizzi, S.; Pasquato, L.; Scrimin, P. J. Am. Chem. Soc. 2005,
127, 1616-1617. (b) Pengo, P.; Broxterman, Q. B.; Kaptein, B.; Pasquato,
L.; Scrimin, P. Langmuir 2003, 19, 2521-2524. (c) Templeton, A. C.;
Hostetler, M. J.; Warmoth, E. K.; Chen, S.; Hartshorn, C. M.; Krishna-
murthy, V. M.; Forbes, M. D. E.; Murray, R. W. J. Am. Chem. Soc.
1998, 120, 4845-4849. (d) Schaaff, T. G.; Knight, G.; Shafigullin, M.
N.; Borkman, R. F.; Whetten, R. L. J. Phys. Chem. B 1998, 102,
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13572-12573. (b) Hong, R.; Fischer, N. O.; Verma, A.; Goodman, C. M.;
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Fischer, N. O.; Verma, A.; Goodman, C. M.; Simard, J. M.; Rotello, V.
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Inhibition of
R
-Chymotrypsin with Nanoparticles
A R T I C L E S
Scheme 1. Synthesis of L-Amino Acid-Terminated Thiol Ligands
Scheme 2. Fabrication of L-Amino Acid-Functionalized Gold Nanoparticles through Place-Exchange Reaction
the nonspecific protein adsorption and preserve the secondary
structure of adsorbed protein.17b,22 In this context, trityl protected
26-mercapto-3,6,9,12,15-pentaoxahexacosan-1-oic acid 1 was
prepared according to Houseman and Mrksich’s protocol23 and
was used as the starting compound for the preparation of amino
acid-functionalized thiol ligands. As shown in Scheme 1, two
methods have been employed to incorporate amino acids into
the thiol ligands. In one method, target thiol ligands 4 were
synthesized by the reaction of 1 with L-amino acid tert-butyl
esters in dry dichloromethane through the activation of car-
boxylic acid function by dicyclohexylcarbodiimide (DCC),
followed by removal of trityl and tert-butyl groups in trifluo-
roacetic acid (TFA) with triisopropylsilane as a hydride donor.24
Alternatively, the coupling reaction of 1 with N-hydroxysuc-
cinimide afforded corresponding active ester, which was further
reacted with free L-amino acids in a mixed solvent of THF and
H2O and successively treated with TFA to give the amino acid
ligands 4 (see Supporting Information).
Once the amino acid-functionalized alkanethiols were ob-
tained, they were subjected to ligand exchange reaction25 with
1-pentanethiol-coated gold nanoparticle (C5-NP, d ≈ 2.0 nm).26
While C5-NP is highly soluble in dichloromethane, the ligand
exchanged nanoparticles were fully precipitated from the
solution. Thus, the nanoparticles were conveniently collected
by centrifugation. All of the resultant nanoparticles show good
solubility in water. Scheme 2 illustrates the fabrication of amino
1
acid-decorated gold nanoparticles and their structures. In H
NMR spectra of the nanoparticles, no signals from the methyl
groups in C5 ligands are observed, indicating that the ligand
exchange is achieved essentially quantitatively. As Murray and
co-workers’ research has demonstrated that place-exchange
proceeds in a 1:1 stoichiometry,25 ∼100 amino acid ligands are
estimated to be present on our 2 nm core particles.25
(22) (a) Zheng, M.; Davidson, F.; Huang, X. J. Am. Chem. Soc. 2003, 125,
7790-7791. (b) Herrwerth, S.; Eck, W.; Reinhardt, S.; Grunze, M. J. Am.
Chem. Soc. 2003, 125, 9359-9366. (c) Kane, R. S.; Deschatelets, P.;
Whitesides, G. M. Langmuir 2003, 19, 2388-2391. (d) Harris, J. M., Ed.
Poly(ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications;
Plenum Press: New York, 1992.
(23) Houseman, B. T.; Mrksich, M. J. Org. Chem. 1998, 63, 7552-7555.
(24) Greene, T. W.; Wuts, P. G. M. ProtectiVe Groups in Organic Synthesis,
3rd ed.; John Wiley & Sons: New York, 1999.
(25) (a) Hostetler, M. J.; Templeton, A. C.; Murray, R. W. Langmuir 1999, 15,
3782-3789. (b) Hostetler, M. J.; Green, S. J.; Stokes, J. J.; Murray, R. W.
J. Am. Chem. Soc. 1996, 118, 4212-4213.
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Soc., Chem. Commun. 1994, 801-802.
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You et al.
Figure 2. Normalized activity of ChT (3.2 µM) with nanoparticles (0.8
µM) bearing various amino acid side chains.
Figure 3. Nonlinear least-squares curve-fitting analysis of the activity assay
data ([ChT] ) 3.2 µM).
ChT Activity Assays and Binding Affinity. Because the
ChT active site is surrounded by cationic residues, negatively
charged nanoparticles can block the active site and subsequently
diminish the accessibility of negatively charged substrates to
the catalytic center, resulting in the inhibition of ChT activity.17
To probe the interaction between ChT and the amino acid-
terminated gold nanoparticles, the ChT-catalyzed hydrolysis of
N-succinyl-L-phenylalanine p-nitroanilide (SPNA) was first
examined in the presence of various concentrations of nano-
particles. The activities of ChT in the presence of nanoparticles
were normalized to that of free ChT.
Table 1. Microscopic Binding Constant (KS), Gibbs Free Energy
Change (-∆G), and Binding Ratio (n) for the Complexation of ChT
with Nanoparticles in Phosphate Buffer (5 mM, pH 7.4) at 30 °Ca
1
1
nanoparticle
KS/106 M-
−∆G/kJ mol-
n
NP_Gly
2.9
7.8
2.7
2.2
3.0
2.4
5.9
13.1
7.1
8.1
37.5
40.0
37.3
36.8
37.6
37.0
39.3
41.3
39.8
40.1
7.3 ( 1.5
6.2 ( 0.2
6.7 ( 0.9
13.1 ( 0.3
6.8 ( 0.5
9.1 ( 0.7
6.4 ( 0.4
6.4 ( 0.2
6.8 ( 0.7
7.6 ( 0.2
NP_L-Ala
NP_L-Asn
NP_L-Asp
NP_L-Gln
NP_L-Glu
NP_L-Leu
NP_L-Met
NP_L-Phe
NP_L-Val
The results show that the nanoparticles exhibit quite a
different influence on the activity of ChT. For most amino acid-
decorated gold nanoparticles, the rate of ChT-catalyzed hy-
drolysis of SPNA decreased upon addition of nanoparticles,
clearly suggesting the activity inhibition through complex
formation (Figure 2). The only exception is L-Arg-terminated
gold nanoparticle, where slight superactivity of ChT was
observed in the presence of NP_L-Arg, indicating that no
significant interaction exists between ChT and NP_L-Arg.17d
Circular dichroism and fluorescence studies gave the same
conclusion (vide post). All of the other hydrophobic, neutral
polar, or negatively charged amino acids-decorated gold nano-
particles show considerable inhibition of ChT activity, indicating
that the complementary electrostatic interaction is essential for
the formation of ChT-NP complexes.
From Figure 2, it is apparent that inhibition of ChT activity
depends critically on the side chain properties of nanoparticles.
While no inhibition effect was observed for NP_L-Arg, the
nanoparticles with polar side chains, for example, NP_L-Asp,
NP_L-Asn, and NP_L-Glu, showed the strongest inhibitory
potency with around 80% ChT activity suppression, while the
nanoparticles with hydrophobic side chains exhibited less
pronounced inhibition. NP_L-Met and NP_L-Ala, for instance,
showed only 60% inhibition on ChT activity. This observation
can be attributed to the hydrophobicity difference of ChT active
site in the presence of different nanoparticles. Presumably, the
hydrophobic active site of ChT is more accessible by SPNA in
comparison with that surrounded by nanoparticles bearing polar
side chains, because the hydrophobic phenylalanine residue in
the substrate needs first to be bound into the active site during
the enzyme-promoted cleavage of peptide bonds.17a As a
consequence, more residual activity of ChT was detected for
a The error of binding constants was usually less than 20%. The
magnitude of binding strength was further confirmed by fluorescence
titration study on the system of ChT and NP_L-Leu, which gave a binding
constant of 5.3 × 106 M-1 (see Supporting Information).
the interaction with nanoparticles carrying hydrophobic side
chains.
The activity assay results were further analyzed to evaluate
the association strength between ChT and nanoparticles. As-
suming that nanoparticles have a number of identical and
independent binding sites (i.e., n) that could bind a ChT
molecule each, the microscopic binding constants (KS) and
binding ratios (n) can be simultaneously estimated by using
nonlinear least-squares curve-fitting analysis on the data of
activity assays (see Supporting Information). Representative
fitting curves are shown in Figure 3, in which no serious
deviations are observed. The excellent curve fits indicate the
reliability of the stability constants. The binding constants and
binding ratios are summarized in Table 1.
From Table 1, it can be seen that the binding constants are
also dependent on the side chains of the nanoparticles. For
nanoparticles with hydrophilic amino acid side chains, that is,
NP_L-Asp, NP_L-Glu, NP_L-Asn, and NP_L-Gln, the binding
constants are around 3 × 106 M-1, comparable to that of
NP_Gly. For nanoparticles with hydrophobic amino acid side
chains, considerably enhanced binding affinity is observed. For
NP_L-Met, the highest binding constant of 1.3 × 107 M-1 is
obtained, which is 4 times that for reference nanoparticle
NP_Gly. These results expressly indicate that the hydrophobic
side chain aids the interaction between nanoparticles and ChT.
A plot of the Gibbs free energy changes (-∆G) for the
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Inhibition of
R
-Chymotrypsin with Nanoparticles
A R T I C L E S
Figure 4. Correlation between Gibbs free energy changes and hydropho-
bicity index of amino acid side chains; represents linear fit of the data.
complexation of ChT with various nanoparticles versus the
hydrophobicity index27 of amino acid side chains shows the
correlation between the binding strength and hydrophobicity of
the side chains (Figure 4).
Gel Electrophoresis and Binding Ratio. It should be noted
from Table 1 that most nanoparticles have binding stoichiom-
etries as determined through activity assays of ∼7, except for
NP_L-Asp and NP_L-Glu that have considerably higher values
of 13 and 9, respectively. To confirm the binding ratios, gel
electrophoresis with various ChT/NP ratios was performed. As
can be seen from Figure 5, the gel electrophoresis results
unambiguously supported the formation of ChT-NP complexes
through charge complementarity. The pre-stain and post-stain
in Figure 5 refer to the treatment of gels before and after staining
of the protein with Comassie Blue, allowing us to observe the
brown nanoparticles before staining and visualize the protein
post-staining. The narrow bands observed for the ChT-NP
complex suggest the formation of discrete complexes rather than
extended ChT-NP aggregates. The maximum positive move-
ment for the particle in the pre-stained gel provides a means
for estimating binding stoichiometries. These studies indicate
that the binding stoichiometry of NP_L-Asn and ChT is 1:7,
while the value for NP_L-Asp and ChT is 1:12. These results
agree well with the corresponding values elucidated from the
activity assays. The gel electrophoresis studies on the other
nanoparticle/ChT systems also gave results consistent with
activity assays, with binding stoichiometries for monotopic
amino acid-functionalized nanoparticles around 1:7, while the
value for NP_L-Glu was 1:10.
Figure 5. Gel electrophoresis of ChT and NP_L-Asn (a) and NP_L-Asp
(b). Nanoparticle concentrations were varied at a constant ChT concentration
(50 µM).
in comparison with NP_MUA by assuming regular spherical
topology. If the binding stoichiometry is critically dependent
on the area of nanoparticle surface, then all of the nanoparticles
should have a binding ratio of 1:12 based on complete coverage
of the nanoparticle surface by ChT. This is not the case,
suggesting that except for NP_L-Asp and NP_L-Glu the
nanoparticle surface is not fully saturated. From the viewpoints
of structure, the number of carboxylates in ditopic amino acid-
functionalized nanoparticles (e.g., NP_L-Asp) should be twice
that of monotopic amino acid-functionalized nanoparticles (e.g.,
NP_L-Asn) if they have the same number of ligands on the
gold core. Thus, it seems that the surface charge density also
affects the binding ratio. X-ray single crystal analysis revealed
that 12 positively charged residues (L-Lys and L-Arg) are located
on the same side of ChT surface and surround the active site
(Figure 1),28 which can take part in the electrostatic interaction
with nanoparticles. On the other hand, it has been demonstrated
that roughly 100 ligands are attached to the Au core in 2 nm
Our previous study revealed that complete inhibition of ChT
using 11-mercaptoundencanoic acid capped gold nanoparticle
(NP_MUA, d ) 5.5 nm) could be accomplished at a NP:ChT
binding ratio of 1:4.17c,d Molecular modeling showed that the
dimension of amino acid-functionalized thiol ligands is around
4 nm, so that the diameters of current nanoparticles are
significantly larger than NP_MUA by 4.5 nm (2 nm Au core
+ ∼8 nm for the ligand chain). Therefore, it is estimated that
the surface area of the current nanoparticles increases ca. 3-fold
(27) Monera, O. D.; Sereda, T. J.; Zhou, N. E.; Kay, C. M.; Hodges, R. S. J.
Pept. Sci. 1995, 1, 319-329. This hydrophobicity index is normalized so
that the most hydrophobic residue (L-Phe) is given a value of 100 relative
to glycine, which is considered neutral (value 0). The scales are extrapolated
to residues that are more hydrophilic than glycine.
(28) (a) Tsukada, H.; Blow, D. M. J. Mol. Biol. 1985, 184, 703-711. (b) Blow,
D. M. Acc. Chem. Res. 1976, 9, 145-152.
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Table 2. Zeta-Potentials for Various Amino Acid-Functionalized
Gold Nanoparticles in PBS Buffer (20 mM potassium phosphate
and 100 mM sodium chloride, pH 7.8) at 25.0 °C
nanoparticle
zeta-potential/mV
nanoparticle
zeta-potential/mV
NP_Gly
-25.8 ( 1.5
-24.7 ( 2.1
-26.2 ( 0.5
-32.5 ( 0.4
-23.5 ( 1.2
NP_L-Glu
NP_L-Leu
NP_L-Met
NP_L-Phe
NP_L-Val
-32.2 ( 0.7
-25.6 ( 1.5
-25.4 ( 2.0
-25.7 ( 1.8
-26.4 ( 1.6
NP_L-Ala
NP_L-Asn
NP_L-Asp
NP_L-Gln
gold nanoparticles.25 Therefore, if the electrostatic interaction
takes place in a 1:1 negative/positive charge ratio or ion pair
form, NP_L-Asn might bind 8 ChT molecules, while NP_L-
Asp has the capacity of associating 16 ChT molecules. These
values are quite close to the observed binding stoichiometries,
suggesting that complete charge pairing is a prerequisite for
efficient binding.
As place-exchange proceeds in a 1:1 stoichiometry25 and we
have complete (by NMR) exchange of the C5 ligands, the charge
on the ditopic particles (i.e., NP_L-Asp and NP_L-Asp) is
expected to be twice that of the monotopic analogues. Qualita-
tive evidence of this expected surface charge disparity of these
nanoparticles was obtained by measuring the zeta-potentials of
particles in PBS buffer at 25.0 °C (Table 2.) As can be seen,
all monotopic amino acid-functionalized nanoparticles have zeta-
potentials of about -25 mV, while the ditopic amino acid-
functionalized nanoparticles such as NP_L-Asp and NP_L-Glu
afford zeta-potentials of around -32 mV. Although the relation-
ship between zeta-potential and charge is not straightforward
due to the complex relationship between surface structure and
measured values,29 these results indicate that the nanoparticles
bearing ditopic amino acid functions possess more negatively
charged surfaces.
Figure 6. Schematic illustration for the binding modes of ChT with
nanoparticles bearing (a) monotopic and (b) ditopic amino acid functions.
In the former case, the surface carboxylate functions are proposed to
reorganize to maximize the electrostatic interactions.
In this context, one rational explanation for the binding ratio
difference between mono- and ditopic amino acid-functionalized
nanoparticles is that the flexible nanoparticle surfaces would
adopt allosteric conformations to achieve the maximal amount
of electrostatic interactions during the complexation process
(Figure 6). Consequently, the surface of nanoparticles with
monotopic amino acids is less saturated than that of NP_L-Asp
or NP_L-Glu by the protein, because the formers have fewer
recognition elements (i.e., carboxylates) on the surface. The
charge-complementary interaction between ChT and NP is
therefore not only dependent on the relative surface areas, but
also regard to the number of charged “hot spots”.
Circular Dichroism. Another important issue to be addressed
is the influence of amino acid side chains on the conformation
of ChT. No CD signals are detected from the nanoparticles,
indicating that the amino acids on the surface are not organized
to an ordered assembly. Figure 7 illustrates the CD spectra of
ChT and ChT with different nanoparticles after 24 h incubation.
It is significant that the CD spectra of ChT are drastically
dependent upon the amino acid side chains of nanoparticles.
While almost no change in the CD spectrum of ChT was
observed with NP_L-Leu, nanoparticles with L-Asn and L-Asp
side chains induced varying levels of CD spectral changes of
ChT, that is, the diminishing of the characteristic minimum at
232 nm and the hypochromic shift of the minimum at 204 nm.
Systematic study showed that the CD signals of ChT are little
Figure 7. Circular dichroism spectra of ChT (3.2 µM) and ChT with
nanoparticles (0.8 µM) after 24 h incubation. The weak CD signal of the
nanoparticles has been subtracted from that of the complex.
influenced by the presence of nanoparticles bearing hydrophobic
amino acid side chains such as NP_L-Phe, NP_L-Leu, NP_L-
Met, NP_L-Val, and NP_L-Ala, while ChT experiences con-
formational changes in the presence of nanoparticles bearing
polar amino acid side chains, for example, NP_Gly, NP_L-Gln,
NP_L-Asn, NP_L-Glu, and NP_L-Asp. The spectral changes
were time-dependent, suggesting that the polar amino acid side
chains induced the conformational changes of ChT, whereas
the hydrophobic amino acid side chains had little effect on ChT
structure. As expected, NP_L-Arg does not perturb the CD
spectrum of ChT.
Fluorescence and Denaturation Kinetics. To obtain insight
into the kinetics of the denaturation process of ChT in the
presence of nanoparticles, the characteristic fluorescence of
tryptophan residues in ChT was investigated using steady-state
fluorescence spectrometry, which is more sensitive than CD.
As shown in Figure 8, the intrinsic emission of ChT shows a
maximum at 331 nm in buffer solution. However, significant
bathochromic shifts and peak broadening were observed after
(29) Kirby, B. J.; Hasselbrink, E. F., Jr. Electrophoresis 2004, 25, 187-202.
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12878 J. AM. CHEM. SOC. VOL. 127, NO. 37, 2005

Inhibition of
R
-Chymotrypsin with Nanoparticles
A R T I C L E S
Figure 9. Linear plots for the first-order chymotrypsin denaturation in the
presence of different nanoparticles in 5 mM sodium phosphate buffer (pH
7.4). The amount of native R-chymotrypsin was calculated from the
fluorescence shifts by assuming that the shifts are proportional to the
denatured species.
Figure 8. Fluorescence spectra of ChT (3.2 µM) and ChT with nanopar-
ticles (0.8 µM) after 24 h incubation.
incubation with nanoparticles for 24 h. The maximal fluores-
cence shift of 23 nm was observed for the ChT incubated with
NP_L-Asp, with concomitant broadening of half-maximal
amplitude from 52 to 68 nm. These phenomena indicate that
the initially buried tryptophan residues were exposed to more
polar environment due to the denaturation of the protein.30 The
extent of denaturation of ChT is reflected by the different values
of fluorescence shift, which is again linked to the properties of
amino acid side chains in nanoparticles. If we consider that the
fluorescence maximum at 356 nm (i.e., L-Trp in water)
corresponds to a complete denaturation of ChT, we may roughly
estimate the extent of protein denaturation by nanoparticles. For
example, after 24 h incubation, hydrophilic NP_L-Asp and
NP_L-Asn induce ca. 90% and 40% denaturation of ChT,
respectively, while hydrophobic NP_L-Phe and NP_L-Leu
induce only 10% and 20% denaturation.
Table 3. First-Order Rate Constants for the Denaturation of ChT
in the Presence of Various Nanoparticles and the Fluorescence
Shifts after Incubation for 24 h
1
nanoparticles
k/10-6 M-1 s-
∆λem/nm
NP_Gly
5.79
3.73
5.33
18.92
4.08
10.18
3.31
0.61
0.41
3.27
11.4
6.2
9.4
23.0
8.2
13.4
5.0
3.2
2.2
5.8
NP_L-Ala
NP_L-Asn
NP_L-Asp
NP_L-Gln
NP_L-Glu
NP_L-Leu
NP_L-Met
NP_L-Phe
NP_L-Val
carboxylate functions facilitate the denaturation process. As
pointed out previously, the competitive hydrogen-bonding
formation may destabilize the R-helices in ChT and disrupt their
secondary structure. Furthermore, these phenomena are most
probably attributed to the fact that the salt bridge (i.e., between
the N-terminus of Ile16 and the Asp194 side chain) in the protein
is more easily broken by the charged side chains. Indeed, the
observed denaturation rates for NP_L-Asp and NP_L-Glu are
comparable to the denaturation rate of ChT at pH 11.0 (i.e., k
) 1.43 × 10-5 M-1 s-1),31 where hydroxide ions are believed
to be involved in the breakage of salt bridges.32
In contrast to the above observations, the hydrophobic amino
acid side chains induced little change in ChT secondary
structure. NP_L-Phe, the nanoparticle bearing the most hydro-
phobic side chain, gives a denaturation rate constant of only
4.1 × 10-7 M-1 s-1, decreasing by 50 times in comparison with
NP_L-Asp. Both CD and fluorescence studies have revealed
that the hydrophobicity of amino acid side chains shows impacts
on the denaturation of ChT. Therefore, the denaturation rate
constants of ChT in diverse nanoparticles were again plotted
against the hydrophobicity index27 of amino acid side chains in
Figure 10. This plot shows that the dianionic side chains strongly
increase the rate of denaturation. The other side chains show a
The denaturation of ChT should occur in a two-state process
as described in eq 1:
ChT-N
9
k8 ChT-D
(1)
where ChT-N is the native form and ChT-D the denatured form,
and k denotes the rate constant. In principle, the denaturation
should follow a first-order reaction profile.
In Figure 9, the logarithm of native ChT concentration was
plotted against time (over a period of 10 h). Good linear
regressions were found for all systems, unambiguously confirm-
ing the first-order denaturation profiles. Interestingly, the rate
of ChT denaturation decreases with the increase of the hydro-
phobicity of amino acid side chains. The rate constants obtained
by the linear regression together with the fluorescence shifts
after 24 h incubation are listed in Table 3.
From Table 3, it can be seen that the denaturation rates are
in accordance with the extent of final fluorescence shifts after
24 h incubation. As expected, NP_L-Asp induces the fast
denaturation of ChT with a rate constant of 1.89 × 10-5 M-1
s-1. For NP_L-Asn, the rate constant drops by two-thirds to
5.33 × 10-6 M-1 s-1. Similarly, for the L-Glu/L-Gln counter-
parts, the rate constants decrease from 1.02 × 10-5 to 4.08 ×
10-6 M-1 s-1. These observations strongly suggest that the
(31) Wu, H.-L.; Lace, D. A.; Bender, M. L. Proc. Natl. Acad. Sci. U.S.A. 1981,
78, 4118-4119.
(32) Wroblowski, B.; D´ıaz, J. F.; Schlitter, J.; Engelborghs, Y. Protein Eng.
1997, 10, 1163-1174.
(30) Ladokhin, A. S. In Encyclopedia of Analytical Chemistry; Meyers, R. A.,
Ed.; John Wiley & Sons Ltd.: Chichester, U.K., 2000; pp 5762-5779.
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A R T I C L E S
You et al.
scaffolds offer opportunities for developing novel core/shell
receptors with highly specific protein-surface recognition ability,
which would possess promising applications in protein stabiliza-
tion, alteration, and delivery.
Experimental Section
General. R-Chymotrypsin (type II from bovine pancreas, ChT),
N-succinyl-L-phenylalanine p-nitroanilide (SPNA), and tert-butyl esters
of amino acids (Gly, L-Phe, L-Ala, L-Val, L-Leu, L-Met, and L-Glu)
were purchased from Sigma and used as received. All of the other
chemicals were obtained from Aldrich unless otherwise stated. The
solvents used for chemical synthesis were purified and dried according
to standard procedures.34 Trityl protected acid 1 was prepared according
to the procedure described by Houseman and Mrksich.23 Subsequently,
trityl protected acid 1 was reacted either with tert-butyl esters of amino
acids or successively with N-hydroxysuccinimide and free amino acids,
followed by the treatment with trifluoroacetic acid and triisopropylsi-
lane, affording corresponding mercaptoligands bearing amino acid
terminals. The purification of all intermediates and ligands was
performed on flash chromatography (SiO2, particle size 0.032-063
mm). Pentanethiol capped gold nanoparticle (d ≈ 2 nm) was prepared
according to the reported method.26 Ligand exchange in dichlo-
romethane was carried out to produce amino acid-decorated gold
nanoparticles (NPs).25 The reaction details and the characterization of
products were compiled in the Supporting Information.
Activity Assays. All of the experiments were performed in sodium
phosphate buffer (5 mM, pH 7.4) with [ChT] ) 3.2 µM and variant
NP concentrations. The enzymatic hydrolysis reaction was initiated by
adding a SPNA stock solution (16 µL) in ethanol to a preincubated
ChT-NP solution (184 µL) to reach a final SPNA concentration of 2
mM. Enzyme activity was followed by monitoring product formation
every 15 s for 15 min at 405 nm with a microplate reader (EL808IU,
Bio-Tek Instruments, Winooski, VT). Control experiment showed that
the autohydrolysis of SPNA was negligible during the time course of
activity assay. The assays were performed in duplicates or triplicates,
and the averages are reported. The standard deviation was usually less
than 10%.
Gel Electrophoresis. Agarose gels were prepared in 5 mM sodium
phosphate buffer at 1% final agarose concentration. Appropriately sized
wells (40 µL) were generated by placing a comb in the center of the
gel. A ChT stock solution of 100 µM in 5 mM sodium phosphate buffer
(pH 7.4) was used to prepare 30 µL samples at the appropriate ChT/
NP ratios ([ChT] ) 50 mM). After a 30 min incubation period at room
temperature, 3 µL of 80% glycerol was added to ensure proper well
loading (30 µL), and a constant voltage (100 V) was applied for 30
min for sufficient separation. Gels were placed in staining solution
(0.5% Coomassie blue, 40% methanol, 10% acetic acid aqueous
solution) for 1 h, followed by extensive destaining (40% methanol,
10% acetic acid aqueous solution) until protein bands were clear. Gels
were scanned on a flatbed scanner both prior to and after staining to
separately visualize particle and ChT bands.
Zeta-Potential. Amino acid-functionalized gold nanoparticles were
dissolved in PBS buffer (20 mM potassium phosphate and 100 mM
sodium chloride, pH 7.8) to make a 1 µM solution, and their zeta-
potentials were measured on a MALVERN Zetasizer Nano ZS
instrument. Three rounds of assays have been performed, and the
average values were reported.
Figure 10. Correlation between the denaturation rate constants (k) of ChT
and the hydrophobicity index of amino acid side chains in nanoparticles.
lesser but still evident correlation between hydrophobicity and
denaturation rate, indicating that the hydrophobicity of amino
acid side chains in nanoparticles plays a role in ChT denaturation/
stabilization.
Our previous study has shown that the alkyl interior in
NP_MUA induces the drastic denaturation of ChT,17c,d whereas
the current hydrophobic amino acid side has little effect on the
native structure of the bound ChT. One plausible interpretation
for these phenomena is that in the former case the alkyl chain
nonspecifically interacts with the whole surface of ChT and
therefore destabilizes the protein. In the latter, however, the
hydrophobic amino acid side chains exclusively interact with
the hydrophobic patches on the ChT surface and preserve the
protein structure much as in the natural protein-protein
interaction. Indeed, selective introduction of hydrophobic groups
near the hydrophobic region on protein surface has proven to
be one method of choice for improving protein stabilization.33
Conclusions
In summary, we have demonstrated that the functionality of
monolayer protected gold nanoparticles could be readily tailored
by introducing diverse terminal functions. By incorporating
simple L-amino acids, nanoparticles exhibit distinctly different
inhibition ability on ChT activity. Complementary electrostatic
interaction between nanoparticles and ChT was proven to be
the predominant driving force contributing to the complex
formation, but the hydrophobic interaction between the hydro-
phobic patches of receptors and proteins enhanced the complex
stability. The binding capacity of these receptors depends not
only on the surface areas of nanoparticles, but also on their
surface charge density. Additionally, the hydrophobicity of
amino acid side chains shows great influence on the secondary
structure of ChT. The hydrophobic side chains have little effect
on ChT structure, while the hydrophilic side chains destabilize
the protein. Taken together, the control over association/
dissociation as well as stabilization/denaturation of ChT at the
MMPC interfaces has been conveniently accomplished by
introducing additional functions in the vicinity of carboxylic
acid functions. These fundamental insights into the interaction
between proteins and artificial receptors with nanoparticle
Circular Dichroism. Far-UV circular dichroism (CD) spectra of
ChT (3.2 µM) were measured on a JASCO J-720 spectropolarimeter
with quartz cuvettes of 1 mm path length at 20 °C. The spectra were
recorded from 190 to 250 nm as an average of three scans at a rate of
20 nm/min. The concentration of NPs is 0.8 µM, and their CD spectra
were subtracted to eliminate background effects.
(34) Perrin, D. D.; Armarego, W. L. F. Purification of Laboratory Chemicals,
(33) Chotia, C. Annu. ReV. Biochem. 1984, 53, 537-572.
3rd ed.; Pergamon Press: Oxford, 1988.
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12880 J. AM. CHEM. SOC. VOL. 127, NO. 37, 2005

Inhibition of
R
-Chymotrypsin with Nanoparticles
A R T I C L E S
Fluorescence. Fluorescence spectra were measured in a conventional
quartz cuvette (10 × 10 × 40 mm) on a Shimadzu RF-5301 PC
spectrofluorophotometer at room temperature (ca. 20 °C). The samples
were excitated at 295 nm, and the emission spectra were recorded from
300 to 450 nm. For the denaturation study, the sample concentrations
are the same as those in the CD study.
Supporting Information Available: Synthesis and charac-
terization of compounds 2, 3, and 4, 1H NMR spectra of
nanoparticles, and the algorithm for the estimation of binding
constants from activity assays. This material is available free
of charge via the Internet at http://pubs.acs.org.
Acknowledgment. This research was supported by the
JA0512881
National Institutes of Health (GM 59249).
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