Catalytic Activity of Peptide-Nanoparticle Conjugates Regulated by a Conformational Change

Article abstract of DOI:10.1021/acs.biomac.7b00887

Herein, we present the design and synthesis of a catalytically active peptide-nanoparticle conjugate whose activity is regulated by a defined conformational change in the self-assembled peptide monolayer. A catalytically active peptide, designed after the

Full text of DOI:10.1021/acs.biomac.7b00887

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Article
Catalytic activity of peptide-nanoparticle
conjugates regulated by a conformational change
Dorian Jamal Mikolajczak, Jason Lee Heier, Boris Schade, and Beate Koksch
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00887 • Publication Date (Web): 19 Sep 2017
Downloaded from http://pubs.acs.org on September 20, 2017
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Catalytic activity of peptideꢀnanoparticle conjugates
regulated by a conformational change
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Dorian J. Mikolajczak , Jason L. Heier , Boris Schade , Beate Koksch *
1
Freie Universität Berlin Department of Biology, Chemistry and Pharmacy, Institute of
Chemistry and Biochemistry, Berlin, Germany
2
Freie Universität Berlin Department of Biology, Chemistry and Pharmacy, Institute of
Chemistry and Biochemistry Research Center for Electron Microscopy, Berlin, Germany
Keywords: catalysis, peptide, gold nanoparticles, selfꢀassembly, nanozymes, regulation
ABSTRACT
Herein, we present the design and synthesis of a catalytically active peptideꢀnanoparticle
conjugate whose activity is regulated by a defined conformational change in the selfꢀassembled
peptide monolayer. A catalytically active peptide, designed after the heterodimeric αꢀhelical
coiledꢀcoil principle was immobilized onto gold nanoparticles and kinetic studies were
performed according to the MichaelisꢀMenten model. The formed peptide monolayer at the gold
nanoparticle surface accelerated pꢀnitrophenylacetate (pNPA) hydrolysis by one order of
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magnitude compared to the soluble peptide while exhibiting no defined secondary structure as
determined by infrared (IR) and circular dichroism (CD) spectroscopy. Addition of the
complementary peptide induced coiledꢀcoil formation while significantly hindering the pNPA
hydrolysis catalyzed by the peptideꢀnanoparticle conjugate. The heptad repeat sequence of a
coiledꢀcoil opens up the opportunity for regulation of conformation and thus catalytic activity of
peptideꢀnanoparticle conjugates upon interaction with a complementary coiledꢀcoil sequence.
Strategies of regulation of catalytic activity by interaction with a complementary cofactor/ligand
are wellꢀestablished in nature and are introduced here into rationally designed peptideꢀ
nanoparticle conjugates.
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INTRODUCTION
The design and creation of nanomolecular systems that mimic the catalytic specificity and
efficiency of enzymes have been a great scientific and technological challenge for decades.
Intensive research has been promoted to elucidate structural features and complex interactions of
1,2
enzymes responsible for their unique catalytic and regulatory mechanisms in order to develop
catalysts with fineꢀtuned specificity, high turnover rates, and increased robustness, that are also
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easily prepared and applied.
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–6
Among different approaches used to create artificial enzymes,
the application of gold
nanoparticles (AuꢀNP) conjugated to a monolayer of thiols (AuꢀMPCs = monolayer protected
7–9
gold clusters) has proven to be a powerful tool. In particular, AuꢀNPs functionalized with a
monolayer of cysteineꢀcontaining peptides exhibit exceptional properties in molecular
10,11
recognition and catalysis.
The high peptide density surrounding the nanoparticle generates
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new microenvironments, which are able to establish new hydrogen bond networks and
cooperative effects between residues that are absent when the peptide or peptides are free in
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solution. As a result, such effects are capable of creating complex environments resembling the
active sites of native enzymes. The application of nanoparticles can therefore yield catalytic
efficiencies increased by several orders of magnitude over nonꢀconjugated peptide variants, and
even generate new catalytic activities, operative under conditions where the monomeric peptide
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shows no catalytic activity.
In addition, the monolayer can be designed to further modulate
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activity by accommodating the lipophilicity of chosen substrate.
The usage of peptide capped AuꢀMPCs had been “exploited to study confinement and clustering
effects on the reactivity of functional groups” in an effort to deepen the understanding of
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complex interactions of amino acid residues located in catalytic sites. To broaden the
knowledge of peptideꢀcapped nanoparticles as a design principle for enzyme mimics, we
investigated the peptide structureꢀfunction relationship of such systems by means of defined
conformational changes and their effect on catalytic activity.
In nature, the catalytic activity of an enzyme is achieved and regulated by its overall protein
topology with solvation of particular residues specifically located within its active site. In other
words, catalytic activity depends on the appropriate folding of the polypeptide chains into
defined threeꢀdimensional structures under specific environmental conditions. In an opposite
way, catalytic activity of peptide capped AuꢀMPCs can be manipulated by forming defined
structures within the peptide monolayer. We herein report the design and synthesis of a peptideꢀ
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nanoparticle conjugate with esterase properties, whose activity is regulated by a defined
conformational change of the peptide monolayer from random into coiledꢀcoil structures.
MATERIALS AND METHODS
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Peptide synthesis
Peptides were manually synthesized on TGA resin using standard fluorenylmethyloxycarbonyl
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(
Fmoc) methodology. The coupling procedure of each Fmocꢀprotected amino acid (5 eq.) was
performed twice employing reagents N,N’ꢀdiisoproplycarbodiimide (DIC) (4.9 eq.) and 1ꢀ
hydroxyꢀ7ꢀazabenzotriazole (HOAt) (5 eq.) in DMF. Sideꢀchain deprotection and peptide/resin
cleavage were accomplished using a cocktail of thioanisole, phenol, triisopropylsilane (TIS), 1,2ꢀ
ethanedithiol (EDT), H O and trifluoroacetic acid (TFA) (5:5:5.2.5:2.5:80, v/v) for peptides
2
containing cysteine and a cocktail of TIS, H O and TFA (3:2:95, v/v) for peptides without
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cysteine. Crude peptides were precipitated in iceꢀcold diethyl ether and purified via reverseꢀ
phase HPLC LaPrep∑ lowꢀpressure HPLC system (VWR, Darmstadt, Germany) using a Kinetex
®
RPꢀC18 endcapped HPLCꢀcolumn (5 µM, 100 Å, 250 x 21.2 mm, Phenomenex , USA). A
TM
®
SecurityGuard PREP Cartridge Holder Kit (21.20 mm, ID, Phenomenex , USA) served as
®
preꢀcolumn. As eluents, deionized water (MilliꢀQ Advantage A10 Ultrapure Water Purification
®
System, Millipore , Billerica, MA, USA) and acetonitrile (ACN), both containing 0.1 % (v/v)
TFA were used. HPLC runs were performed starting with an isocratic gradient of 5 % ACN over
5 min, flow rate: 10 mL/min, continuing with a linear gradient of 5 – 70 % ACN over 25 min,
flow rate: 20.0 mL/min.
Nanoparticle synthesis
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−2
An aqueous solution of HAuCl (0.2 mL, 2.49 • 10 M) was added to 2 mL of enzymeꢀfree
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water (EFꢀH O, Water for Molecular Biology, Merck Millipore). Under vigorous stirring,
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.5 mL of aqueous peptide E3H15 (5.438 • 10 M) solution and 0.03 mL of 1 M NaOH solution
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were added. Following 30 min of stirring, 0.1 mL aqueous 0.1 M NaBH was added and a wine
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red dispersion was immediately formed. After stirring for an additional 60 min, the dispersion
was centrifuged using Amicon Ultraꢀ0.5 centrifugal filters (NMWL 30kDa) to recover a pellet of
peptideꢀcapped gold nanoparticles. The pellet was redispersed in EFꢀH O and again centrifuged
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to remove excess NaBH . The resulting pellet was redispersed in EFꢀH O and stored at room
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temperature. Nanoparticle concentration was calculated according to equations provided by Liu
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et al. An average loading of 265 peptides per gold nanoparticle was determined by analyzing
the centrifuged supernatants by UV/Visꢀspectroscopy using a Cary 50 UV/Visꢀspectrometer
(Varian) and reverseꢀphase HPLC. The amount of bound E3H15 was obtained by subtracting the
amount of unbound peptide present in the supernatant from the amount of peptide initially used
in the synthesis.
Peptide stock solutions
Peptide stock solutions were prepared by dissolving lyophilized peptide in 50 mM Tris/HCl
buffer pH 7.3. Concentrations of stock solutions were determined by measuring the UVꢀlight
absorbed by a diluted aliquot. Applying the LambertꢀBeer law and taking into account dilution
factors, concentrations of peptide stock solutions were calculated according to their respective
ꢀ1
ꢀ1
chromophore. For E3H15, an extinction coefficient (ε ) of 1263 L mol cm was applied,
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which was experimentally determined by measuring the absorbance of tyrosine in H Nꢀ
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CGGYEꢀOH at 280 nm
in 50 mM Tris/HCl pH 7.3. For the peptide K3, which contains the
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chromophore 2ꢀaminobenzoic acid (Abz), a preꢀdetermined extinction coefficient (ε ) of H Nꢀ
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ꢀ1
ꢀ1
AbzꢀGlyꢀOH at 320 nm of 2980 L mol cm was applied.
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Michaelis-Menten kinetic assay
MichaelisꢀMenten kinetics was applied to assess the rate of hydrolysis of the ester substrate
pNPA at pH 7.3 (50 mM Tris/HCl) and 25°C. Reactions were monitored by measuring the
absorbance of the formed product pꢀnitrophenolate at 405 nm. Using an experimentally
ꢀ1
ꢀ1
determined extinction coefficient of 9919 L mol cm , initial rates of reaction (v ) of
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uncatalyzed (blank) and catalyzed reactions were determined over a range of substrate
concentrations until saturation conditions were observed. Initial rates of reaction of the catalyst
were obtained by subtracting v of the blank reaction from v of the corresponding catalyzed
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reaction. All kinetic experiments were performed at least three times to determine standard
deviation. The maximum rate of reaction at saturating substrate concentrations vmax and the
Michaelis constant K which is the substrate concentration at which the rate of the catalyzed
M,
reaction is half vmax, were obtained by performing a nonꢀlinear regression of the Michaelisꢀ
Menten equation. Errors were calculated according to error propagation derived from the
Gaussian equation for normally distributed errors.
All reactions were performed in quartz QS cuvettes with a 1 cm pathlength and, equipped with
Teflon stoppers (3.5 mL; Hellma Analytics) in a UV/Visꢀspectrophotometer (Cary 50, Varian)
thermostatted at 25° C (Lauda RKS). All solutions used in the reactions were allowed to
equilibrate at 25 °C 20 minutes prior to the start of the reaction. These solutions included a stock
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solution of pNPA dissolved in acetonitrile as well as the reaction cuvette containing only buffer
(blank) or peptide dissolved in buffer. Reactions were initiated by adding a volume of substrate
solution so the total volume of all reaction mixtures contained 2% acetonitrile. The total solution
volume of each reaction was 1 mL.
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Kinetic assays with E3H15 were performed in 50 mM Tris/HCl buffer pH 7.3 at 25 °C with a
peptide concentration of 50 µM. In these assays, initial pNPA concentrations of 0.1, 0.2, 0.5, 1.0,
2.0 and 4.0 mM in the reaction mixture were employed.
Kinetic assays with the synthesized peptideꢀgold nanoparticle conjugate (Au@E3H15) as well as
measurements containing 5 µM K3 were performed in 50 mM Tris/HCl buffer pH 7.3 at 25 °C
using a calculated concentration of 5 µM immobilized E3H15 peptide. Initial pNPA
concentrations of 0.05, 0.075, 0.1, 0.2, 0.3 and 0.5 mM were applied in these reactions.
Circular dichroism (CD) spectroscopy
CDꢀspectra were obtained using a Jasco Jꢀ810 spectropolarimeter with a HAAKE WKL
recirculating chiller (Dꢀ76227, Karlsruhe). The cuvettes were set to a constant temperature of
25° C with a Jasco PTCꢀ423S Peltierꢀtype thermocouple. Measurements were performed in
quartz cuvettes (Suprasil Hellma) having a pathlength of 2 mm.
FT-IR spectroscopy
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ꢀ1
IRꢀspectra were recorded using an ATRꢀFT/IRꢀ4100 by Jasco, averaging 100 scans with 3 cm
resolution. Lyophilized peptide and peptideꢀgold nanoparticle conjugates were placed on the
diamond crystal. IRꢀspectra were recorded, taking into account the corresponding background.
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Transmission electron microscopy (TEM)
Aliquots (5 µL) of the aqueous samples were applied to a formvar/carbon covered copper grid
(
400 mesh, PLANO GmbH, Wetzlar) which was hydrophilized by 60 s glow discharging at 8 W
in a BALTEC MED 020 device directly before use. After 30 s of AuNP sedimentation excess
liquid was removed with filter paper and the grid was allowed to airꢀdry for at least 20 min. The
grid was then transferred to a Tecnai F20 transmission electron microscope (FEI Company,
Oregon) equipped with a field emission gun operating at an acceleration voltage of 160 kV.
Image data were recorded using a FEI Eagle 4k × 4k CCD camera with a primary magnification
of 100,000 x resulting in an image resolution of 0.214 nm/dot of the electron micrographs.
Nanoparticle size and size distribution were determined using ImageJ (Image processing and
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analysis in JAVA) V 1.50 by analyzing at least 1000 particles.
RESULTS AND DISCUSSION
Peptide design
Peptides E3H15 and K3 (Fig. 1) were designed according to the wellꢀcharacterized IAALꢀ
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0
E3/IAALꢀK3 coiledꢀcoil peptide system. Both peptides include the basic principles of a parallel
heterodimeric αꢀhelical coiledꢀcoil folding motif. Thus, by combining both peptides, coiledꢀcoil
formation is readily induced.
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Figure 1. Peptide sequences of E3H15 and K3. Heptad repeat patterns are highlighted in blue
(E3H15) and orange (K3) and represented in the helical wheel diagram.
The peptide E3H15 was designed to feature a histidine residue in position 15 among several
glutamate residues. Thus, an imidazole moiety is present in a carboxylateꢀrich environment to
create a catalytically active chargeꢀrelay network capable of accelerating ester hydrolysis.
Furthermore, the generation of cooperative carboxylateꢀimidazole interactions is likely enhanced
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when peptides are immobilized as has previously been reported. Covalent attachment of E3H15
onto the gold nanoparticle surface is easily provided by a thiolꢀgold bond via the Nꢀterminal
2
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cysteine residue.
Preparation and characterization of Au@E3H15
Preparation and functionalization of the gold nanoparticles were performed by reduction of an
Au(III) salt by NaBH in presence of E3H15, which also acts as a stabilizing ligand. The average
4
size of gold nanoparticles was determined by transmission electron microscopy (TEM).
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Concentration of gold nanoparticle dispersion was calculated according to equations reported by
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Liu et al. (Supporting Information). TEM revealed spherical Au@E3H15 particles with an
average diameter of 5.4 ± 2.0 nm (Fig. 2).
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Figure 2. TEMꢀimage of Au@E3H15.
By taking into account the amount of bound peptide as determined by supernatant analysis, the
average peptide loading was determined to be 265 peptides per gold nanoparticle.
To confirm peptide binding and to detect changes in the peptide IR spectrum owing to the
immobilization process, FTꢀIR spectroscopy was performed (Fig. 3 A). FTꢀIR showed the
presence of IR stretching frequencies of multiple bands present in E3H15, such as Amide A,
ꢀ1
Amide I and Amide II. Most strikingly, the Amide A band at 3289 cm appears to be
significantly broader for Au@E3H15 than for E3H15. This broadening of the Amide A band
indicates multiple new hydrogen bond interactions of the NꢀH groups likely resulting from the
increased peptide density at the nanoparticle surface and a close proximity between
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intermolecular residues. The inability of E3H15 and Au@E3H15 to form defined secondary
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structures under the specified conditions was validated by CD spectroscopy (Fig. 3 B), which
resulted in spectra having single ellipticity minima at 199 and 191 nm, respectively.
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Figure 3. (A) FTꢀIR spectra of E3H15 and Au@E3H15. (B) Normalized CD spectra of E3H15
and Au@E3H15. CD spectra were recorded at pH 7.3 and 25 °C in 50 mM Tris/HClꢀbuffer with
2
% acetonitrile and a total peptide concentration of 50 µM.
Effect of immobilization on catalytic efficiency
In the present study, catalyzed ester hydrolysis was accessed using the chromogenic model ester
pNPA. This ester serves as a benchmark substrate for studying the hydrolytic activity of
2
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enzymes and designed peptide catalysts. The dependence of the initial rates of pNPA
hydrolysis on pNPA concentration is in accordance with the MichaelisꢀMenten model for E3H15
and Au@E3H15. However, due to its low catalytic activity, a minimum concentration of 50 µM
E3H15 was needed to clearly observe catalysis. On the other hand, a lower peptide concentration
of 5 µM of immobilized E3H15 provided sufficient study of Au@E3H15ꢀcatalyzed hydrolysis
while avoiding experimental complications caused by the highly absorbent nature of AuꢀNPs.
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Table 1. Kinetic parameters kcat, K and kcat/K of pNPA hydrolysis at 25 °C in Tris/HCl
M
M
buffer pH 7.3 with 2 % acetonitrile in the presence of 50 µM E3H15 and K3 for E3H15 and
E3H15 / K3 and 5 µM immobilized E3H15 and K3 for Au@E3H15 and Au@E3H15 / K3.
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Catalyst
E3H15
kcat [10 s ]
K [mM]
k /K [M s ]
cat M
M
1.92 ± 0.25
2.29 ± 0.51
7.20 ± 1.09
ꢀ
0.98 ± 0.26
1.49 ± 0.56
0.31 ± 0.09
ꢀ
0.20 ± 0.08
0.15 ± 0.09
2.18 ± 0.95
ꢀ
E3H15 / K3
Au@E3H15
Au@E3H15 / K3
Table 1 shows concentrationꢀindependent kinetic parameters k , K and k /K . The
cat
M
cat
M
immobilization of E3H15 onto gold nanoparticles led to an 11ꢀfold increase in catalytic
efficiency k /K . Interestingly, the improvement of catalytic efficiency originates from a
cat
M
decrease in K and an increase in turnover number of the catalyst k , both by a factor of
M
cat
approximately three. A decrease in K indicates an increased binding affinity of pNPA for the
M
peptide monolayer of Au@E3H15 compared to the free peptide. This finding could be explained
with the formation of transient enzymeꢀlike binding pockets at the Au@E3H15 surface in which
the substrate is preferentially bound. Riccardi et al. validated the existence of such cavities
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7
within selfꢀorganized monolayers of thiolꢀfunctionalized gold nanoparticles. They showed that
AuꢀNPs functionalized with oligo(ethylene glycol)ꢀbased thiols are able to bind and
accommodate small organic aromatic molecules such as salicylate in selfꢀorganized transient
binding pockets within the monolayer with different selectivity and sensitivity similar to proteinꢀ
ligand recognition. We speculate that such binding processes could also occur within the peptide
monolayer of Au@E3H15, leading to the observed decrease in KM.
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The increase in turnover number k , which is also the first order rate constant of Au@E3H15
cat
shows that the hydrolytic process in the Au@E3H15 monolayer occurs more rapidly. This can be
attributed to an additionally formed cooperative carboxylateꢀimidazolium mechanism, as
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proposed by Pengo et al.
The free peptide containing the amphoteric histidine imidazole and
carboxylate groups is expected to follow a catalytic general acid and general base mechanism
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(
Scheme 1A) similar to His/Glu or His/Asp dyads in esterases. In case of Au@E3H15, the high
local peptide density surrounding the nanoparticle and thus presentation of multiple carboxylate
moieties in close proximity to the imidazole can lead to an increase in its pK , thus favoring the
a
protonated imidazolium ion. The imidazolium ion, in turn, acts as a general acid, forming a
stabilizing hydrogen bond with the developing charge of the subsequent tetrahedral transition
state of ester hydrolysis. In this scenario, the tetrahedral transition state is initiated by a
carboxylate anion, acting as a general base. This additional cooperative hydrolytic pathway is
said to be absent in the monomeric peptideꢀsolution and results in a rate acceleration of the
1
2,13
hydrolytic process.
Another mechanism for the rate acceleration of nucleophilic reactions on
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carbonyl compounds catalyzed by imidazolium cations was proposed by Ullah et al. Thereby,
the imidazolium cation interacts with the carbonyl oxygen, activating the carbonyl carbon and
facilitating the following nucleophilic attack. Based on the mechanistic proposals we assume the
mechanism of pNPA hydrolysis catalyzed by Au@E3H15 to be as depicted in Scheme 1B.
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Scheme 1. (A) Proposed mechanism for the hydrolysis of pNPA catalyzed by carboxylate anions
and imidazole present in E3H15. (B) Proposed mechanism for the hydrolysis of pNPA catalyzed
by carboxylate anions and imidazolium cations additionally present in the Au@E3H15
monolayer.
Regulation of activity by coiled-coil formation
Having determined the catalytic activity of Au@E3H15, we investigated the effect
conformational change has on catalytic activity. By themselves, Au@E3H15 and K3 each
showed no defined secondary structures (Fig. 4A). Au@E3H15 when mixed with equimolar K3
resulted in increased αꢀhelical content as observed by CD spectra showing two new minima at
222 nm and 202 nm. By virtue of the chosen peptide design, coiledꢀcoil conformation is likely
induced on interaction of E3H15 and K3 and adopted.
Kinetic studies performed on Au@E3H15 in the presence of equimolar K3 revealed an average
decrease in catalytic activity of Au@E3H15 by 85 % at all substrate concentrations (Fig. 4B). In
contrast, a marginal decrease in catalytic activity was observed for soluble coiledꢀcoil E3H15 in
the presence of K3 (Fig. 4C, D).
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Figure 4. (A) Normalized CD spectra of Au@E3H15, K3 and a mixture of both. (B) Michaelisꢀ
Menten plots of blankꢀcorrected pNPA hydrolysis catalyzed by Au@E3H15 and
Au@E3H15/K3. (C) Normalized CD spectra of E3H15, K3 and a mixture of both. (D)
MichaelisꢀMenten plots of blankꢀcorrected pNPA hydrolysis catalyzed by E3H15 and
E3H15/K3. Error bars depict standard deviations of at least three independent measurements.
The effective reduction of catalytic efficiency due to interaction with complementary inhibitor
K3 in case of Au@E3H15 is most likely due to a combination of several effects. The E3H15
chains are involved in more rigid αꢀhelical coiledꢀcoil structures which hinders the formation of
possible binding cavities. The fixed orientation of histidine and glutamate residues within a
coiledꢀcoil arrangement leads to reduction of intramolecular cooperative interactions necessary
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for substrate activation and stabilization. Furthermore, intercalation of K3 into the E3H15
monolayer can disrupt intermolecular cooperative residue interactions responsible for the
increased catalytic efficiency of Au@E3H15. The presence of lysineꢀrich K3 within the
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glutamateꢀrich E3H15 monolayer could also lead to a decreased intrinsic pk of the histidine
a
imidazole moiety by electrostatic effects as opposed to the solely E3H15 monolayer. In either
case, the additional cooperative mechanism is diminished and catalysis is decreased. Detailed
mechanistic studies of Au@E3H15ꢀcatalyzed ester hydrolysis and its inhibition by K3 are
currently ongoing.
Conclusion
Herein, we report the first example of a peptideꢀnanoparticle conjugate whose catalytic activity is
regulated by a defined change in peptide conformation from random coil to coiledꢀcoil
structures. The immobilization of peptide E3H15 onto spherical AuꢀNPs (Au@E3H15) triggers
cooperative glutamateꢀhistidine interactions resulting in an increase in catalytic efficiency of
pNPA hydrolysis by more than one order of magnitude in comparison to nonꢀimmobilized
E3H15. Au@E3H15 shows no defined secondary structure as was validated by IR and CD
spectroscopy. However, when combined with complementary coiledꢀcoil peptide K3 in
equimolar ratios, coiledꢀcoil formation was induced. The generation of rigid coiledꢀcoil
structures and the formation of a mixed layer of E3H15 and K3 led to a significant inhibition of
catalytic activity. Detailed mechanistic studies of the complex interactions within these peptide
monolayers are ongoing.
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Our results show the importance peptide topology has on the catalytic activity of peptideꢀ
nanoparticle conjugates and present a viable approach towards the design of enzyme mimics
with more complex structureꢀfunction relationships.
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ASSOCIATED CONTENT
Supporting Information
UV/Visꢀspectroscopic measurements showing absorbance against time spectra of pNPA
hydrolysis at different concentrations catalyzed by Au@E3H15. UV/visꢀspectrum of
Au@E3H15. Calibration curve of pNPA blank reactions. Comparison between UV/visꢀspectra of
H NꢀCGGYEꢀOH dilution series and supernatant of Au@E3H15 after synthesis. RPꢀHPLC
2
chromatogram of Au@E3H15 supernatant after synthesis. Equations used to determine gold
nanoparticle concentrations.
AUTHOR INFORMATION
Corresponding Author
Beate Koksch, Freie Universität Berlin
Department of Biology, Chemistry, Pharmacy Institute of Chemistry and Biochemistry Berlin,
Germany.
Email: beate.koksch@fuꢀberlin.de
Funding Sources
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The authors gratefully thank the Integrated Research Training Group (Graduiertenkolleg)
“Multivalency in Chemistry and Biochemistry” of the Collaborative Research Center 765 (SFB
765) “Multivalency as Chemical Organizational and Operational Principle: New Architectures,
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Functions, and Applications” at the Freie Universitaet Berlin funded by the German Research
Foundation (DFG).
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