Contrasting effects of exterior and interior hydrophobic moieties in the complexation of amino acid functionalized gold clusters with α-chymotrypsin

Article abstract of DOI:10.1021/ol052367k

(Figure Presented) A series of L-amino acid functionalized gold nanoparticles with oligo(ethylene glycol) (OEG) tethers of varying length are prepared. These studies show that the hydrophobic side chains of amino acids facilitate the structural retention

Full text of DOI:10.1021/ol052367k

ORGANIC
LETTERS
2005
Vol. 7, No. 25
5685-5688
Contrasting Effects of Exterior and
Interior Hydrophobic Moieties in the
Complexation of Amino Acid
Functionalized Gold Clusters with
r
-Chymotrypsin
Chang-Cheng You, Mrinmoy De, and Vincent M. Rotello*
Department of Chemistry, UniVersity of Massachusetts, 710 North Pleasant Street,
Amherst, Massachusetts 01003
rotello@chem.umass.edu
Received September 30, 2005
ABSTRACT
A series of
L
-amino acid functionalized gold nanoparticles with oligo(ethylene glycol) (OEG) tethers of varying length are prepared. These
-chymotrypsin (ChT) but the interior alkyl
studies show that the hydrophobic side chains of amino acids facilitate the structural retention of
r
chains promote its denaturation. An 80-fold range of denaturation rate constants were obtained for ChT in the presence of various nanoparticles.
Thus, the tunable denaturation of protein could be achieved by rational combination of amino acid side chains and OEG tethers.
Protein recognition by artificial receptors provides a potent
tool to modulate cellular processes such as signal transduc-
tion, DNA transcription, and protein enzyme inhibition. To
date, a number of synthetic agents such as calixarene
derivatives1 and chemically modified cyclodextrins2 have
been exploited in protein surface recognition and have shown
effective modulation of the structure and function of bio-
macromolecules. Monolayer protected clusters (MPCs) and
mixed monolayer protected clusters (MMPCs) are attractive
artificial receptors for biomacromolecules such as proteins
and DNA.3 The MMPC motif represents an appealing
alternative for generating structurally well-defined bulky
organic functional entities with dimensions comparable to
biomacromolecules, which are often not easily accessible
through conventional organic protocols. Moreover, the
functionality of MMPCs is readily tailored by proper design
of the organic coating molecules.4
(3) For recent reviews, see: (a) Verma, A.; Rotello, V. M. Chem.
Commun. 2005, 303-312. (b) Pasquato, L.; Pengo, P.; Scrimin, P. J. Mater.
Chem. 2004, 14, 3481-3487. (c) Katz, E.; Willner, I. Angew. Chem., Int.
Ed. 2004, 43, 6042-6108. (d) Shenhar R.; Rotello, V. M. Acc. Chem. Res.
2003, 36, 549-561. (e) Sastry, M.; Rao, M.; Ganesh, K. N. Acc. Chem.
Res. 2002, 35, 847-855.
(4) (a) Abad, J. M.; Mertens, S. F. L.; Pita, M.; Fernandez, V. M.;
Schiffrin, D. J. J. Am. Chem. Soc. 2005, 127, 5689-5694. (b) Zheng, M.;
Huang, X. J. Am. Chem. Soc. 2004, 126, 12047-12054. (c) Lin, C.-C.;
Yeh, Y.-C.; Yang, C.-Y.; Chen, G.-F.; Chen, Y.-C.; Wu, Y.-C.; Chen, C.-
C. Chem. Commun. 2003, 2920-2921.
(1) (a) Park, H. S.; Lin, Q.; Hamilton, A. D. J. Am. Chem. Soc. 1999,
121, 8-13. (b) Hamuro, Y.; Calama, M. C.; Park, H. S.; Hamilton, A. D.
Angew. Chem., Int. Ed. Engl. 1997, 36, 2680-2683.
(2) (a) Otzen, D. E.; Knudsen, B. R.; Aachmann, F.; Larsen, K. L.;
Wimmer, R. Protein Sci. 2002, 11, 1779-1787. (b) Leung, D. K.; Yang,
Z. W.; Breslow, R. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5050-5053.
10.1021/ol052367k CCC: $30.25
© 2005 American Chemical Society
Published on Web 11/11/2005

Our recent studies5 revealed that carboxylate-terminated
MMPCs are able to target R-chymotrypsin (ChT), a serine
protease with a positively charged surface (pI ) 8.75) and
well-studied enzymatic activity, through surface comple-
mentary electrostatic interaction. More interestingly, we
found that MMPCs featuring mercaptoundecanoic acid
ligands rapidly denatured the bound ChT molecules, while
the incorporation of additional tetra(ethylene glycol) spacers
onto MMPCs reduced considerably the denaturation extent.
Therefore, it is essential to clarify how the linkages between
recognition elements and MMPC cores affect the protein-
receptor interaction. In this paper, we have synthesized a
family of 30 L-amino acid functionalized MMPCs (Figure
1)6,7 with oligo(ethylene glycol) (OEG) tethers of variable
to afford biocompatible monolayers.8 The varible amino acids
at the periphery of MMPCs allow us to detect the side chain
effect upon interaction with ChT.7
Activity assays were first conducted to evaluate the
inhibitory potencies of these MMPCs on the enzymatic
activity of ChT. With N-succinyl-l-phenylalanine p-nitro-
anilide (SPNA) as substrate, the acitivity of ChT was
drastically depressed upon addition of most MMPCs (Figure
2 for NP_3EG_L-Val). Only L-Arg functionalized MMPCs
Figure 2. Progress curves for the hydrolysis of SPNA in the
presence of ChT and various concentrations of NP_3EG_L-Val.
[ChT] ) 3.2 µM and SPNA [2 mM].11 (Inset) Normalized activity
of ChT in the presence of varying concentrations of NP_3EG_L-
Val.
exhibited no inhibition due to their positively charged side
chains. Consequently, the complementary electrostatic in-
teraction between ChT and MMPCs plays a vital role in the
complex formation.9 The enzymatic activity of ChT typically
decreased ca. 60-85% upon incubation with excess MMPCs
(Figures S24 and S25). The binding strength between ChT
and MMPCs was quantified by analyzing the activity assay
data through nonlinear least-squares curve-fitting using an
equal K model where the MMPCs were assumed to possess
n identical and independent binding sites.7 The results show
that microscopic binding constants for complexations be-
tween ChT and MMPCs are between 106 and 107 M-1 (Table
S1).10 Interestingly, the length of OEG tethers shows
considerable influence on the complex stability of ChT and
MMPCs (Figure 3). For most MMPCs the affinity increases
with decreasing length of OEG tether. One plausible
Figure 1. (a) Structural features of amino acid functionalized
MMPCs and ChT. The residues in red are in the active site, and
residues in blue are cationic. (b) Chemical structure of amino acid
functionalized MMPCs.
length and explored their interaction with ChT. OEG units
are well-known to resist nonspecific interaction with bio-
molecules and have been deposited onto various substrates
(8) (a) Lasseter, T. L.; Clare, B. H.; Abbott, N. L.; Hamers, R. J. J. Am.
Chem. Soc. 2004, 126, 10220-10221. (b) Zheng, M.; Davidson, F.; Huang,
X. J. Am. Chem. Soc. 2003, 125, 7790-7791. (c) Herrwerth, S.; Eck, W.;
Reinhardt, S.; Grunze, M. J. Am. Chem. Soc. 2003, 125, 9359-9366. (d)
Kane, R. S.; Deschatelets, P.; Whitesides, G. M. Langmuir 2003, 19, 2388-
2391. (e) Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical
Applications; Harris, J. M:, Ed.; Plenum Press: New York, 1992.
(9) Control experiments showed that L-Arg-functionalized MMPCs can
inhibit the enzymatic activity of negatively charged â-galactosidase (pI )
4.6). The other MMPCs with neutral or negatively charged side chains
conversely show no inhibition on this enzyme.
(10) The binding affinity of these MMPCs toward ChT is comparable
to the naturally occurring ChT-inhibitor interaction: (a) Chen, C.; Hsu,
C.-H.; Su, N.-Y.; Lin, Y.-C.; Chiou, S.-H.; Wu, S.-H. J. Biol. Chem. 2001,
276, 45079-45087. (b) Fukada, H.; Takahashi, K.; Sturtevant, J. M.
Biochemistry 1985, 24, 5109-5115.
(5) (a) Hong, R.; Fischer, N. O.; Verma, A.; Goodman, C. M.; Emrick,
T.; Rotello, V. M. J. Am. Chem. Soc. 2004, 126, 739-743. (b) Fischer, N.
O.; McIntosh, C. M.; Simard, J. M.; Rotello, V. M. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 5018-5023.
(6) The MMPCs were prepared by place-exchange of 1-pentanethiol-
protected nanoparticles (d ≈ 2 nm) with corresponding amino acid
functionalized thiolate ligands in dichloromethane. 1H NMR spectra show
that the exchange is accomplished quantitatively. It is estimated that about
100 amino acid functionalized ligands are anchored on each nanoparticle:
Hosteler, M. J.; Wingate, J. E.; Zhong, C.; 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.
(7) You, C.-C.; De, M.; Han, G.; Rotello, V. M. J. Am. Chem. Soc. 2005,
127, 12873-12881.
5686
Org. Lett., Vol. 7, No. 25, 2005

Figure 4. CD spectra of ChT (3.2 µM) in the absence and presence
of L-Leu functionalized MMPCs (0.8 µM) with different OEG
tethers in 5 mM sodium phosphate buffer (pH 7.4) after 24 h
incubation.
Figure 3. Binding constants (log KS) between ChT and MMPCs
bearing various amino acid side chains and OEG tethers, estimated
from activity assays.
structure of ChT is efficiently denatured by MMPCs.12
Deconvolution of the circular dichroism spectra revealed that
the contents of R-helices drastically decreased along with
the augmentation of â-sheets.13 The OEG length-related
denaturation indicates that OEG tethers can aid in the
retention of ChT secondary structure. The amino acid side
chains also displayed different levels of influence on the
protein structure. Hydrophilic amino acids such as L-Asp,
L-Glu, L-Asn, and Gly always induced significant CD spectral
changes, whereas hydrophobic amino acids such as L-Phe,
L-Leu, and L-Val showed less pronounced effect. Overall,
the hydrophilic residues facilitate the loss of protein second-
ary structure.
The conformational change of ChT in the presence of
MMPCs was further quantified using fluorescence spectrom-
etry. The steady-state fluorescence maximum of ChT is 331
nm with a half-height width of 54 nm (Figure 5). Upon
incubation with MMPCs for 24 h, significant bathochromic
shifts of fluorescence maxima and peak broadening was
observed. For NP_1EG_L-Val, the fluorescence maximum
was red-shifted to 352 nm (∆λ ) 21 nm) with concomitant
broadening of half-maximal amplitude to 68 nm. These
phenomena indicate that the initially buried tryptophan
residues are exposed to more polar environment as a result
of the unfolding of the protein.14
interpretation for this phenomenon is that the shorter linkers
are more preorganized, decreasing the entropic cost of
binding.
Both activity assays and gel electrophoresis studies showed
that the binding capacity of ditopic amino acid terminated
MMPCs is higher than that of monotopic amino acid
functionalized MMPCs, in accordance with our previous
studies.7 For example, the binding stoichiometries of all four
L-Asp functionalized MMPCs are ca. 11-13, while the
binding stoichiometries of monotopic amino acid (e.g.,
L-Leu) functionalized MMPCs are ca. 6-8. Surprisingly, the
binding capacity is essentially irrespective of the length of
OEG tethers for the tested MMPCs. These results suggest
that the binding ratios are dependent on the surface “hot
spots” (i.e., carboxylates) rather than proportional to the
surface area, which is expected to decrease from 300 to 180
nm2 going from the 4EG to the 1EG spacer. In this context,
the association between ChT and these MMPCs is proposed
to operate through intermolecular ion pairs, and the highly
mobile carboxylates on the MMPC periphery tend to cluster
together to realize the maximal electrostatic interaction with
ChT through cooperative interactions.
Circular dichroism (CD) spectra of ChT with various
MMPCs were subsequently recorded to determine the impact
of amino acid side chains as well as the OEG tethers on the
protein structure. Figure 4 shows the CD spectra of ChT upon
incubation with a series of L-Leu terminated MMPCs for 24
h. Whereas ChT that was incubated with NP_4EG_L-Leu
showed negligible CD spectral changes, the protein displayed
different levels of CD spectral changes upon incubation with
L-Leu MMPCs that bear shorter OEG tethers. Among the
four MMPCs, NP_1EG_L-Leu with the shortest OEG tether
induced the most significant spectral changes, i.e., the
decrease of the characteristic minimum at 230 nm and the
blue shift of the minimum at 204 nm. The spectral changes
were found to be time-dependent (as examplified in Figures
S26-S29). This observation clearly shows that the initial
The denaturation of ChT follows a first-order reaction
profile. In Figure 6, the logarithm of native ChT concentra-
tion is plotted against the incubation period with L-Leu
(11) The absorbance was converted to a concentration scale by a molar
absorption coefficient of 9800 M-1 cm-1 for 4-nitroaniline. Stra¨ter, N.; Sun,
L.; Kantrowitz, E. R.; Lipscomb, W. N. Proc. Natl. Acad. Sci. U.S.A. 1999,
96, 11151-11155.
(12) This denaturation is partially reversible as evidenced by the
enzymatic activity recovery of ChT by addition of sodium chloride into
the system to dissociate the complexes.
(13) Sreerama, N.; Woody, R. W. Anal. Biochem. 2000, 287, 252-260.
The deconvolution using CONTINLL protocol showed that the R-helical
content in ChT decreased from 16.8% to 6.9%, whereas â-sheet content
increased from 30.2% to 36.7% with NP_1EG_L-Leu.
(14) Ladokhin, A. S. In Encyclopedia of Analytical Chemistry; Meyers,
R. A., Ed.; John Wiley & Sons Ltd.: Chichester, U.K., 2000; pp 5762-
5779.
Org. Lett., Vol. 7, No. 25, 2005
5687

Figure 5. Fluorescence spectra of ChT (3.2 µM) in the absence
and presence of L-Val decorated MMPCs with different OEG tethers
in 5 mM sodium phosphate buffer (pH 7.4) after 24 h incubation.
Figure 7. Histograms of first-order denaturation rate constants of
ChT upon incubation with MMPCs bearing various amino acid side
chains and OEG tethers in 5 mM sodium phosphate buffer (pH
7.4).
anionic, promote the denaturation. For the series of 1EG and
2EG, however, drastic denaturation of ChT was observed
for all amino acids. Such results are attributed to the exposure
of the interior alkyl chains to the protein surface, accelerating
the conformational mutation of the protein through nonspe-
cific hydrophobic interaction.15 The denaturation rates of
MMPCs that bear the same amino acid function but different
OEG length all increase in the order 4EG < 3EG < 2EG <
1EG. The spacers of three or more EG units significantly
reduce the nonspecific interaction of proteins with interior
hydrophobic shell and preserve the native structure of
proteins. Therefore, tunable denaturation of proteins can be
accomplished by proper combination of the amino acid
functions and the OEG tethers in MMPCs.
In summary, we have demonstrated that hydrophobic
amino acid side chains favor the retention of the protein
structure. Access to the interior alkyl chains, however,
denatures the protein as a result of nonspecific hydrophobic
interactions. These interactions are essentially eliminated by
introducing tethers with three or more EG units. The tunable
denaturation of proteins is thus achievable by adjusting either
the recognition elements (amino acids) or the OEG linkers.
Figure 6. Linear plots for the first-order ChT denaturation in the
presence of L-Leu terminated MMPCs with different OEG tethers
in 5 mM sodium phosphate buffer (pH 7.4).
functionalized MMPCs. Good linear regressions are obtained,
confirming the first-order reaction (see Supporting Informa-
tion). It can be seen from Figure 6 that the denaturation rates
decrease with the length increase of OEG linkage, i.e., 1EG
> 2EG > 3EG > 4EG, which is in accordance with the CD
study.
The denaturation rate constants of ChT in the presence of
various MMPCs were calculated from their kinetic curves
(Figure 7; see also Table S2). The denaturation rate constants
vary from 4.1 × 10-7 M-1 s-1 for NP_4EG_L-Phe to 3.1 ×
10-5 M-1 s-1 for NP_1EG_L-Asp. For the series of 3EG
and 4EG, the denaturation rates of ChT decrease with
increasing hydrophobicity of the side chains. This trend
suggests again that the hydrophilic side chains, in particular
Acknowledgment. This research was supported by the
National Institutes of Health (GM 59249).
Supporting Information Available: Synthesis and char-
1
acterization of ligands and the preparation and H NMR
spectra of corresponding MMPCs, representative CD spectra,
and plots for the first-order denaturation of ChT. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(15) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115,
10714-10721.
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