Monolayer-controlled substrate selectivity using noncovalent enzyme-nanoparticle conjugates

Article abstract of DOI:10.1021/ja0461163

Electrostatic interactions were used to noncovalently conjugate chymotrypsin to gold nanoparticles featuring hybrid tetraethylene(glycol)alkanethiol monolayers terminated with carboxylate groups. This conjugation process greatly alters the substrate selectivity of the adsorbed chymotrypsin, inhibiting the hydrolysis of anionic subtrates without affecting the hydrolysis rate of cationic analogues. Copyright

Full text of DOI:10.1021/ja0461163

Published on Web 09/29/2004
Monolayer-Controlled Substrate Selectivity Using Noncovalent
Enzyme-Nanoparticle Conjugates
Rui Hong,† Todd Emrick,‡ and Vincent M. Rotello*,†
Department of Chemistry, Department of Polymer Science and Engineering,
UniVersity of Massachusetts Amherst, Amherst, Massachusetts 01003
Received June 30, 2004; E-mail: rotello@chem.umass.edu
Enzyme modification and immobilization have been extensively
studied and utilized to generate biocatalysts with improved stability
and selectivity.1 Enhanced or altered enzyme selectivity not only
affords novel biocatalysts directly but also finds application in
sensing and enzyme-related biotechnology.2 Given the highly
specific interaction between enzymes and substrates, genetic
mutation or chemical modification of enzymes represents the
primary method for alteration of substrate selectivity.3 An alternative
strategy for altering enzyme selectivity, however, is through
noncovalent surface interaction with protein using synthetic scaf-
folds.4 The large surface area and ample surface functionalization
chemistry of nanometer-scale materials, especially monolayer-
protected nanoparticles,5 have made them promising scaffolds to
manipulate enzyme activities.6
Figure 1. Chemical structure of the TCOOH ligand and schematic depiction
of substrate-monolayer interaction-induced enzyme selectivity.
In our previous work,7 we have shown control over chymotrypsin
(ChT) structure and function by using surface-functionalized CdSe
nanoparticles. The interaction between ChT and nanoparticle ligands
is electrostatically driven. Binding of ChT to nanoparticles func-
tionalized with alkanethiol-tetra(ethylene glycol)acetic acid ligands,
TCOOH (Figure 1), inhibits the ChT-catalyzed hydrolysis of
succinyl-Phe-pNA (SPNA) without protein denaturation. The
inhibition was attributed to the spatial blocking of the ChT active
site by the nanoparticle scaffold. However, a substrate-selective
behavior of the protein-nanoparticle complex was observed in
further investigations, which could not be explained by pure steric
effects. We report in this communication that the surface-bound
protein (i) retains activity and (ii) exhibits pronounced substrate
chemoselectivity.
Anionic TCOOH-functionalized Au nanoparticles (Au-TCOOH)
were prepared by standard place exchange chemistry8 (see Sup-
porting Information (SI)). Binding of ChT to Au-TCOOH was
achieved through electrostatic attraction as its CdSe analogue.
Distinguishing our approaches from previously reported protein-
nanoparticle complexes in which the cysteine-gold interaction was
the driving force,3,9 here the interacting sites were built into the
ligand structure, rather than directly on the gold surface. We
consider this an important alternative to control protein-nano-
particle interactions, allowing variation of the surface functionalities
and the reduction of nonspecific interactions.10 The adsorption of
ChT onto Au-TCOOH nanoparticles was confirmed as in our
previous studies7 by activity assays and fluorescence quenching of
Trp residues in ChT (see SI).
Figure 2. Chemical structure of the substrates and the normalized activity
of ChT bound to Au-TCOOH relative to ChT alone with these substrates.
See Supporting Information for activity assay protocol.
degree of relative activity of ChT toward these substrates. With
SPNA, GPNA, and SAPNA, the ChT activity decreased dramati-
cally when bound to Au-TCOOH, with residual activities of ∼15%
for SPNA and below 10% for the other two. When the neutral
substrate BTNA was used, however, a ∼70% relative activity to
free ChT was observed.
One similarity we noted for all the substrates inhibited was the
presence of carboxylic acid groups in the molecules, which gives
negative charge to the substrates in neutral pH buffer. With the
elimination of charges, such as with BTNA, much less inhibitory
effect was observed. Although this observation suggested that
electrostatic repulsion between the carboxylate of the substrate and
the anionic nanoparticle monolayer may contribute to ChT selectiv-
ity, other variables of the substrate structures, such as size and
hydrophobicity, made difficult the direct attribution of the observed
selectivity to electrostatic effects.
To further elucidate the role of substrate charge on the selectivity,
three SPNA derivatives, 1, 2, and 3 with different charges, were
synthesized and analyzed (see SI for synthesis). Heterofunctional-
ized tri(ethylene glycol) (EG3) molecules, with a primary amine
at one end for conjugation to SPNA, and a charged moiety at the
other end, were used to modify SPNA. Carbodiimide coupling
reactions, followed by necessary deprotection, successfully afforded
desired products 1 with carboxylic acid, 2 with alcohol, and 3 with
primary amine end groups in good yields (Figure 3a). The choice
of EG3 as the linker between SPNA and the charged groups ensured
A series of commercially available oligopeptides was tested as
substrates for ChT, including SPNA, N-benzoyl-tyrosine-pNA
(BTNA), glutaryl-Phe-pNA (GPNA), and succinyl-Ala-Phe-pNA
(SAPNA) (Figure 2). The ChT-catalyzed hydrolysis reaction was
followed spectroscopically by monitoring the formation of p-
nitroaniline (pNA). Interestingly, we found a substantially different
† Department of Chemistry.
‡ Department of Polymer Science and Engineering.
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10.1021/ja0461163 CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
ChT-AuTCOOH was negative, thus favoring the adsorption of
cationic 3 to the monolayer. The increased local concentration of
3 near the nanoparticle surface in turn increased the accessibility
of it to the enzyme. The seemingly unchanged activity can thus be
viewed as a cancellation of the unfavorable steric hindrance by
favorable electrostatic attraction.
In summary, we have presented control over enzymatic activity
at a higher level than a simple “on/off” mode. The bound enzyme
retains activity and exhibits enhanced chemoselectivity due to the
substrate-monolayer interactions. Our finding has three major
implications. First, the monolayer of the protein-binding nano-
particle scaffolds can be used to control the interactions between
the protein and nanoparticle. Second, the structure of the monolayer
can also be used to control enzyme-substrate or protein-ligand
interactions; a useful attribute of surface binding that is difficult to
achieve with small molecular recognition units. Finally, the
interactions of protein-substrate-3D nanoparticle monolayer13
studied here also give insight into those interactions on 2D SAMs
or on other solid supports, which is important for the fabrication
of bioactive surfaces and materials.14
Figure 3. (a) Structures of the modified SPNA substrates 1-3. (b) Initial
rates of ChT hydrolysis of these modified substrates. (Inset) Normalized
activity of Au-TCOOH-bound ChT toward substrates 1-3.
sufficient water solubility of the modified substrates, especially for
neutral substrate 2. EG3 also extended the charged groups away
from the binding and catalytic sites of the enzymatic reaction,11
thereby minimizing secondary effects of the modification. Most
importantly, this modification provided molecules of very similar
structure, differing only at the EG3 chain ends. This minimized
the differences in chain length and hydrophobicity and emphasized
the effect of charged groups on the enzymatic activity of surface-
bound ChT.
Acknowledgment. This research was supported by the National
Institutes of Health (GM 62998, V.R.) and a National Science
Foundation Career Award (CHE-0239486, T.E.).
Supporting Information Available: Synthesis of Au-TCOOH and
modified SPNA compounds, activity assay protocol, fluorescence
quenching of ChT by Au-TCOOH, activity of ChT with Au-TCOOH
at elevated ionic strength. This material is available free of charge via
the Internet at http://pubs.acs.org.
Each of the modified SPNA molecules proved to be good sub-
strates for ChT as shown in Figure 3b. No detectable auto-
hydrolysis was observed for any of the modified SPNA compounds.
The higher hydrolysis rate of these modified SPNA molecules
relative to that of SPNA itself confirmed the successful substrate
design and synthesis.12 The initial rates of enzymatic hydrolysis
by native ChT were similar for all the SPNA derivatives, which
directly reflected the similarity in the structures of these molecules.
Enhanced chemoselectivity of ChT was observed when bound
to the Au-TCOOH surface. The ChT-AuTCOOH complex
showed very low activity toward negatively charged substrate 1.
However, a ∼50% and a nearly 100% relative activity of bound
ChT to free ChT were observed toward the neutral substrate 2 and
the positively charged substrate 3, respectively (Figure 3). In a
control experiment, when the activity assays were performed in a
solution of elevated ionic strength (200 mM, with NaCl), at which
condition the electrostatic attraction was screened as evident in
fluorescence assay (see SI), ChT with Au-TCOOH displays almost
identical activity as ChT alone toward all the modified SPNAs (see
SI). Therefore, the observed selectivity can be directly attributed
to binding of ChT to the Au-TCOOH surface monolayer. Con-
sidering the characteristic substrate structure, together with the
anionic nature of the nanoparticle monolayer, this chemoselectivity
can be explained by a combination of steric hindrance and
electrostatic interactions (Figure 1). For negatively charged 1, the
interactions between 1 and the surface-bound ChT were disfavored
by steric and electrostatic effects. As a result, the catalytic reaction
was dramatically slowed, in accord with anionic substrates SPNA,
GPNA, and SAPNA. For neutral substrate 2, steric effects hindered
the substrate hydrolysis, but there were no unfavorable electrostatic
interactions. An intermediate inhibitory effect was then observed;
BTNA was also in this class as the charge repulsion was not present.
In the case of 3, substrate access to ChT was presumably affected
by binding to Au-TCOOH. However, the overall charge of the
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