Peptide conjugates for directing the morphology and assembly of 1D nanoparticle superstructures

Article abstract of DOI:10.1002/chem.201304074

Designed peptide conjugates molecules are used to direct the synthesis and assembly of gold nanoparticles into complex 1D nanoparticle superstructures with various morphologies. Four peptide conjugates, each based on the gold-binding peptide (AYSSGAPPMPPF

Full text of DOI:10.1002/chem.201304074

DOI: 10.1002/chem.201304074
Communication
&
Materials Chemistry
Peptide Conjugates for Directing the Morphology and Assembly
of 1D Nanoparticle Superstructures
Chen Zhang,[a] Chengyi Song,[a] H. Christopher Fry,[b] and Nathaniel L. Rosi*[a]
in the context of constructing and assembling 1D gold nano-
Abstract: Designed peptide conjugates molecules are
particle superstructures.
used to direct the synthesis and assembly of gold nano-
We have developed synthetic methods that utilize peptide
particles into complex 1D nanoparticle superstructures
conjugate molecules to direct the simultaneous synthesis and
with various morphologies. Four peptide conjugates, each
assembly of inorganic nanoparticles.[2] The peptide conjugates
based on the gold-binding peptide (AYSSGAPPMPPF;
consist of a peptide containing a particular sequence of amino
PEPAu), are prepared: C12H23O-AYSSGAPPMPP (1), C12H23O-
acids for binding to a specific inorganic surface[1f,3] and a partic-
AYSSGAPPMPPF (2), C12H23O-AYSSGAPPMPPFF (3), and
ular organic moiety attached to the peptide N-terminus that
C12H23O-AYSSGAPPMPPFFF (4). The affect that C-terminal
influences peptide assembly. We demonstrated that such pep-
hydrophobic F residues have on both the soft-assembly of
tide conjugates can be used to prepare a diverse collection of
the peptide conjugates and the resulting assembly of
complex nanoparticle assemblies containing well-defined
gold nanoparticle superstructures is examined. It is shown
structures with tailorable shapes, metrics, and properties.[2]
that the addition of two C-terminal F residues (3) leads to
One can make numerous modifications to the peptide conju-
thick, branched 1D gold nanoparticle superstructures,
gate molecule (Scheme 1) that may affect its assembly. Much
whereas the addition of three C-terminal F residues (4)
leads to bundling of thin 1D nanoparticle superstructures.
Scheme 1. Illustration of a peptide conjugate detailing the different poten-
tial regions for modification: R=organic tail; Xm and Yn =additional amino
acids; PEPAu =AYSSGAPPMPPF.
Inorganic nanoparticles are important structural and functional
building blocks for the assembly of new advanced materials.[1]
Any given material may have a hierarchy of structural and
functional domains, including, for example, nanoparticles,
nanoparticle superstructures, and finally assemblies of nano-
particle superstructures. Controlling each level of this hierarchy
is essential for controlling material properties and, ultimately,
application. Numerous methods exist for preparing discrete
nanoparticles of various size, shape, and composition,[1a–e] and
the number of viable methods for carefully assembling nano-
particles into nanoparticle superstructures is rapidly gro-
wing.[1f–k] Ideally, nanoparticle assembly methods should
permit control over each level of a material’s structural hierar-
chy: 1) the size, shape, and composition of the nanoparticle
building blocks, 2) the metrics and morphology of the nano-
particle superstructure, and 3) the co-assembly of the nanopar-
ticle superstructures into larger-scale structures. In this Com-
munication, we present assembly methods that address 1–3)
of our work has focused on modifying the N-terminus with
various aliphatic carbon chains or p-conjugated molecules (R
in Scheme 1).[2a,c–e,g] One can also modify the peptide sequence,
provided that these modifications do not alter the peptide’s in-
organic recognition sequence and therefore its inorganic bind-
ing capabilities. Along these lines, we have introduced alanine
residues to the N-terminus (Xm in Scheme 1) of a gold-binding
peptide (PEPAu =AYSSGAPPMPPF)[4] and shown how these addi-
tional amino acids influence peptide conjugate assembly and
ultimately nanoparticle superstructure assembly.[2c,e] When such
conjugates are allowed to assemble in aqueous conditions, the
hydrophobic organic molecule and the amino acids at the N-
terminus are expected to locate in the core of the peptide as-
sembly while the bulk of the peptide and its C-terminus are ex-
pected to locate on the surface of the assembly, exposed to
the aqueous buffer solution.[5] Based on this assembly para-
digm, we predict that modifications to the C-terminus (Yn in
Scheme 1) should significantly affect conjugate assembly and
possibly lead to new nanoparticle superstructures.
[a] C. Zhang, Dr. C. Song, Prof. N. L. Rosi
Department of Chemistry, University of Pittsburgh
219 Parkman Avenue, Pittsburgh, Pennsylvania 15260 (USA)
Fax: (+01)412-624-8611
In our first studies in this area, we demonstrated that C12-
PEPAu (PEPAu containing a C12 chain attached to its N-terminus;
C12 =C12H23O) assembles into 1D twisted fibers and directs the
synthesis and assembly of left-handed gold nanoparticle
double-helical superstructures.[2a] We recently reported that
these helical superstructures exhibit plasmonic circular dichro-
E-mail: nrosi@pitt.edu
[b] Dr. H. C. Fry
Center for Nanoscale Materials, Argonne National Laboratory
9700 South Cass Avenue, Argonne, Illinois 60439 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201304074.
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ism properties,[2f] which could ultimately be useful for the
preparation of chiral metamaterials,[6] provided that individual
double helices could be aggregated together into an extended
array. Our hypothesis in this work is that the addition of extra
phenylalanine (F) residues to the C-terminus of C12-PEPAu could
promote interfibril interactions in buffer that would lead to the
formation of fiber bundles, which could in turn serve to direct
the formation of thick linear nanoparticle superstructures or
possibly even bundles of double-helical superstructures. Thus,
the C-terminus might potentially be used as a handle for con-
trolling the co-assembly of 1D nanoparticle superstructures
into larger scale structures. We note that it is well-established
that hydrophobic F residues can impact peptide assembly in
aqueous conditions,[7] and that F2 alone, for example, assem-
bles into nanotubular structures.[8] However, it has not been
demonstrated that hydrophobic F residues can direct the ag-
gregation or bundling of linear nanoparticle superstructures.
We explore this phenomenon for the first time in this report.
To explore our hypothesis, we designed the following series
of peptide conjugate molecules: C12-AYSSGAPPMPP (1), C12-
AYSSGAPPMPPF (2),[2a] C12-AYSSGAPPMPPFF (3), and C12-AYSS-
GAPPMPPFFF (4). We first studied the assembly of these pep-
tide conjugates in 0.1m HEPES buffer (pH 7.3Æ0.1; HEPES=4-
(2-hydroxyethyl)-piperazineethanesulfonic acid). We dissolved
samples of conjugate 1–4 in HEPES to yield 150 mm peptide
conjugate solutions, which were allowed to sit at room tem-
perature for various specific time periods (30 min, 1, 3, and
7 days). After these time periods, we spotted TEM grids with
samples from each solution, applied a phosphotungstic acid
stain, and then imaged each sample by TEM to monitor the
progress of assembly (Figure 1). Several general conclusions
can be drawn from the micros-
dling for both conjugate 2 and 3 after longer incubation times
in HEPES buffer. Significant bundling for conjugate 3 was first
observed after one day, and many thick bundles (width=
35.7Æ12.4 nm) were observed after one week, as observed by
both TEM (Figure 1) and tapping-mode AFM studies (see Fig-
ure S4 in the Supporting Information). The AFM data reveal
fiber bundles with a height of ~20 nm, which indicates that
these bundles are intertwined fibers rather than thick flat
sheets of individual fibers. Conjugate 2 required a longer incu-
bation time to form bundles: significant fiber bundling was
only observed after three days and after one week the bun-
dling was not nearly as significant as conjugate 3. Conjugate 4,
which contains three terminal F residues, forms fibers most
rapidly, and some of these fibers bundle together; however,
we observe fewer and thinner fiber bundles for 4 than 3. We
reason that since conjugate 4 rapidly assembles into fibers, the
fibers may precipitate from solution before having sufficient
time to interact with each other during assembly to allow for-
mation of large quantities of fiber bundles.
The peptide conjugate assembly studies detailed above
were performed in the absence of gold salt, which is an inte-
gral component of our nanoparticle superstructure synthese-
s.[2a–f] From previous studies by us and others, we know that
Au3+ (as well as other multivalent cations, such as Ca2+) can
significantly accelerate and affect peptide conjugate assem-
bly.[2a,9] It is known from these studies that the multivalent cat-
ions shield the negatively charged regions of the peptide, pre-
venting interpeptide electrostatic repulsions and promoting
conjugate assembly.[9] In our system, once gold salts are added
to solutions of peptide conjugates in HEPES, fiber growth rap-
idly occurs and gold nanoparticles form and assemble along
copy data. First, after a short in-
cubation period (30 min), no as-
sembly is observed for conjugate
1, while conjugates 2 and 3
both assemble into spherical
structures (diameter of 2=
21.3Æ3.4 nm, based on 100
counts; diameter of 3=21.0Æ
3.8 nm, based on 100 counts),
and conjugate 4 assembles into
1D fibers (width=6.5Æ0.8 nm,
based on 100 counts; see Fig-
ure S3 in the Supporting Infor-
mation for distribution diagrams;
note: all reported distributions
are based on measurements
from TEM images of multiple
samples). With increasing time,
conjugates 1–4 all form fibers.
For each conjugate, we observe
an increasing number of fibers
with increased incubation time,
and also with an increasing
Figure 1. TEM images of soft assemblies prepared using peptide conjugates 1, 2, 3, or 4 in HEPES buffer at differ-
ent time points (0.5 h, 1 d, 3 d, 7 d). White arrows indicate points where individual or thin fibers merge to form
number of C-terminal F residues.
Finally, we observed fiber bun- thick fiber bundles.
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the fibers to yield gold nanoparticle superstructures, because
HEPES can reduce Au3+ to Au0.[4,10] The rapid formation of gold
nanoparticle superstructures prevents careful observation of
conjugate assembly. To study conjugate assembly under condi-
tions that more accurately reflect the conditions used to pre-
pare the gold nanoparticle superstructures, we investigated
peptide conjugate assembly in the presence of calcium cat-
ions. Specifically, we added 0.1m calcium chloride (1.5 mL,
0.15 mmol) to 150 mm solutions of conjugate 1–4 in 125 mL
0.1m HEPES (Figure S5 in the Supporting Information). After in-
cubating for 30 min at room temperature with added Ca2+
,
conjugates 1–4 each formed fibers. The degree of fiber forma-
tion increased with an increasing number of C-terminal F resi-
dues and, again, some bundling was observed for conjugates
2 and 3, with conjugate 3 exhibiting much more significant
bundling than 2. These results mirror what we observed after
seven day incubation without Ca2+, which suggests that in-
creasing the ionic strength by adding Ca2+ accelerates the as-
sembly process. We anticipated that assembly of conjugates
1–4 in the presence of Au3+ would be similar to their assembly
Figure 3. Magnified TEM images of nanoparticle superstructures formed
using peptide conjugates 1 (a), 2 (b), 3 (c), and 4 (d).
in the presence of Ca2+
.
After having established that the addition of C-terminal F
residues significantly affects fiber assembly and bundling, we
next proceeded to explore the impact of these modifications
on nanoparticle superstructure formation in the presence of
gold salt. For these studies, we followed our established syn-
thetic protocol.[2a] Chloroauric acid solution (1.0 mL, 0.1m
HAuCl4 in 1.0m TEAA; TEAA=triethylammonium acetate) was
added to a solution of peptide conjugate (1–4) in 0.1m HEPES
buffer. After incubating these solutions for one day at room
temperature, the reaction products were imaged by using TEM
(Figures 2, 3 and Figures S6–9 in the Supporting Information).
Conjugate 1 yielded aggregates of 1D gold nanoparticle super-
structures (Figures 2a, 3a, and Figure S6 in the Supporting In-
formation, width: 24.1Æ4.1 nm, based on 100 counts; nano-
particle diameter: 8.1Æ1.3 nm, based on 100 counts), while
conjugate 2 yielded well-defined gold nanoparticle double
helices, as previously reported (Figure 2b, 3b, and Figure S7 in
the Supporting Information, width: 20.1Æ1.8 nm, based on
100 counts; nanoparticle diameter: 7.3Æ1.4 nm, based on 100
counts). Conjugate 3 yielded thick, branched 1D nanoparticle
superstructures (Figure 2c, 3c and Figure S8 in the Supporting
Information, width: 61.4Æ15.7 nm, based on 100 counts;
nanoparticle diameter: 6.3Æ1.2 nm, based on 100 counts).
Conjugate 4 resulted in thin intertwined 1D nanoparticle su-
perstructures (Figure 2d, 3d, and Figure S9 in the Supporting
Information, width: 20.5Æ4.9 nm, based on 100 counts; nano-
particle diameter: 6.0Æ1.1 nm, based on 100 counts). These re-
sults are illustrated and summarized in Scheme 2.
The results from these studies are generally consistent with
our peptide conjugate assembly studies. Superstructures
formed by using conjugate 1 are linear, yet very irregular (Fig-
ure 2a and Figure S6 in the Supporting Information). We note
that in this case, the terminal F residue of PEPAu has been de-
leted; this may negatively affect the formation of discrete
nanoparticles and lead to irregular or aggregated nanoparti-
Scheme 2. The number of Fx residues (x=0–3) at the C-terminus of C12-
AYSSGAPPMPPFx determines the structure of 1D gold nanoparticle super-
structures. x=0: aggregated 1D structures; x=1: double helices; x=2:
thick, branched 1D structures; x=3: thin, intertwined structures.
Figure 2. TEM images of nanoparticle superstructures formed using peptide
conjugates 1 (a), 2 (b), 3 (c), and 4 (d).
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cles. In fact, previous studies have demonstrated that the ter-
minal F residue of PEPAu is important for binding to the Au sur-
face.[11] Conjugate 3 predominantly forms thick bundles of
fibers, which leads to the formation of thicker 1D nanoparticle
assemblies (Figure 2c and Figure S8 in the Supporting Informa-
tion). In this case, we collected images of superstructure for-
mation after 50 min of reaction (see Figure S10 in the Support-
ing Information). It is clear that multiple individual fibers
bundle together to form thick fibers, and we can observe
nanoparticle growth on these thick bundles. At this stage, it is
unclear why the nanoparticles only decorate the fiber bundles
rather than individual fibers; we are currently investigating the
mechanism of superstructure formation using conjugate 3,
which may provide insight into this observation.
ticle superstructures. These results indicate that C-terminal
modifications to peptide conjugates represent another catego-
ry of synthetically addressable features that can be carefully
tuned to impact the structure of nanoparticle superstructures
and the co-assembly of these structures. Finally, these studies,
when grouped with our previous studies on these systems,[2]
demonstrate that peptide conjugates are a highly tailorable
class of molecules whose structures and compositions can be
carefully programmed to direct the synthesis and assembly of
a diverse array of complex hierarchical nanoparticle-based
structures.
Experimental Section
We observe some important similarities between the nano-
particle superstructure products of conjugate 2 and 4 (Fig-
ure 2b, d, and Figures S7 and S9 in the Supporting Informa-
tion). Consistent with our previous studies,[2a,b,f] conjugate 2
predominantly assembles into nonaggregated fibers, which
lead ultimately to the formation of isolated double-helical
nanoparticle assemblies as the major product and some bun-
dled and intertwined double helices. Conjugate 4 forms a mix-
ture of fibers and fiber bundles, which leads to the formation
of some isolated yet a majority of intertwined 1D nanoparticle
superstructures. The widths of these thin nanoparticle super-
structures (2: 20.1Æ1.8 nm; and 4: 20.5Æ4.9 nm) are consis-
tent with having a single fiber (2: 6.7Æ0.9 nm; and 4: 6.5Æ
0.8 nm) decorated with nanoparticles (2: 7.3Æ1.4 nm; and 4:
6.0Æ1.1 nm). The nanoparticle arrangement in the superstruc-
tures formed with conjugate 4 is difficult to discern, although
we can identify some regions of the structure that appear to
exhibit a helical arrangement of the nanoparticles (arrows in
Figure S9, Supporting Information). Superstructures from con-
jugate 4 assemble together to form larger-scale structures with
diameters ranging from ~20 to ~60 nm (see Figure S9 in the
Supporting Information). These data indicate that the addition
of F residues (in the case of conjugate 4) provides a way to as-
semble helical nanoparticle assemblies into larger-scale struc-
tures.
Materials and methods
All solvents and chemicals were obtained from commercial sources
and used without further purification. 0.1m HEPES Buffer (HEPES=
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) was made by
directly diluting 1.0m HEPES buffer (pH 7.3Æ0.1; Fisher Scientific)
with water (NANOpure, Barnstead DiamondTM System; 18.2 MW).
Peptides with sequences of AYSSGAPPMPP, AYSSGAPPMPPF, AYSS-
GAPPMPPFF, and AYSSGAPPMPPFFF were synthesized and purified
by New England Peptide with final purity of 99%. AYSSGAPPMPPFE
was prepared by Argonne National Laboratory with final purity of
99%. Reverse-phase HPLC was performed at ambient temperature
with an Agilent 1200 liquid chromatographic system equipped
with diode array and multiple wavelength detectors using a Grace
Vydac protein C4 column (214TP1010, 1.0 cmꢁ25 cm). Matrix-as-
sisted laser desorption ionization time-of-flight (MALDI-TOF) mass
spectra were obtained on an Applied Biosystem Voyager System
6174 MALDI-TOF mass spectrometer using a-cyano-4-hydroxy cin-
namic acid (CHCA) as the matrix. TEM samples were prepared by
pipetting one drop of solution onto a 3 mm-diameter copper grid
coated with carbon film; 2% aqueous phosphotungstic acid was
used for negative staining. TEM was conducted on either a JEOL
200CX instrument operated at 200 kV and equipped with a Gatan
CCD image system or FEI Morgagni TEM operated at 80 kV and
equipped with an AMT side mount CCD camera system. Samples
for atomic force microscopy (AFM) were prepared on freshly
peeled MICA substrates. Tapping-mode AFM was performed on
a Veeco Dimension V SPM.
To investigate whether C-terminal F residues are important
for fiber formation and fiber bundling, we studied the assem-
bly of C12-PEPAu-E (5; C12-AYSSGAPPMPPFE). E, glutamic acid, is
hydrophilic. Solutions of 5 (150 mm in 0.1m HEPES buffer) were
allowed to stand for one week. Negatively stained TEM images
of the assembly product revealed only spherical assemblies
(see Figure S11 in the Supporting Information). In addition,
only spherical assemblies were observed after 30 min incuba-
tion in the presence of Ca2+ (see Figure S12 in the Supporting
Information). These data suggest that C-terminal hydrophobici-
ty may be important for directing fiber formation and bun-
dling.
Preparation of N-hydroxyl-succinimide ester and peptide
conjugates
N-Hydroxyl-succinimide ester: Dodecanoic acid (696 mg, 6 mmol)
and N-hydroxysuccinimide (725 mg, 6.3 mmol) were dissolved in
dry ethyl acetate (30 mL) under an argon atmosphere. After addi-
tion of dicyclohexyl carbodiimide (DCC) (1341 mg, 6.5 mmol) at
08C, the solution was stirred overnight at room temperature. The
reaction mixture was processed by removing the precipitate by fil-
tration. The solvent was removed under reduced pressure and the
crystalline residue recrystallized from isopropanol (iPrOH) to yield
the N-hydroxyl-succinimide ester (211 mg, 1 mmol, 17%).
Peptide conjugates: Peptide conjugates (1–5) were synthesized
and purified by using established methods.[2a] Briefly, for conjugate
1, AYSSGAPPMPP (1.20 mg, 8.80ꢁ10À7 mol) was dissolved in DMF
(60 mL). After the addition of dodecanoic N-hydroxyl-succinimide
ester (0.6 mg, 2.81ꢁ10À6 mol) in DMF (60 mL) and Et3N (1 mL) under
stirring, the solution was stirred at room temperature for 16 h.
Pure peptide conjugate 1 was obtained by conducting reversed-
In this study we investigated the impact that modifications
to the C-terminus of C12-PEPAu have on the soft assembly of
peptide conjugates and the structure and assembly of 1D
nanoparticle superstructures. We discovered that the addition
of hydrophobic F residues can lead to fiber bundling that in
turn leads to the formation of thick or intertwined 1D nanopar-
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phase HPLC (see Figure S1 in the Supporting Information) eluting
with a linear gradient of 0.05% formic acid in CH3CN and 0.1%
formic acid in water (5:95 to 95:5 over 30 min). The molecular
weight for each peptide conjugate was confirmed by MALDI-TOF
mass spectrometry (see Figure S2 in the Supporting Infromation).
Concentration of the peptide was determined spectrophotometri-
cally in water/acetonitrile (1:1) using the molar extinction coeffi-
cient of tyrosine (1280mÀ1 cmÀ1) at 280 nm.
Keywords: gold · nanoparticles · peptides · self-assembly ·
superstructures
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Acknowledgements
Support for this work was provided in part by the National Sci-
ence Foundation (DMR-0954380, N.L.R.) and in part by the Air
Force Office of Scientific Research (FA9550-11-1-0275, N.L.R.). A
portion of the peptide synthesis was performed at the Center
for Nanoscale Materials, a U. S. Department of Energy, Office of
Science, and Office of Basic Energy Sciences User Facility under
Contract No. DE-AC02-06CH11357. The authors thank the Pe-
terson NFCF, the MEMS Department and Department of Biolog-
ical Sciences for provision of access to TEM.
Received: October 17, 2013
Published online on December 16, 2013
Chem. Eur. J. 2014, 20, 941 – 945
www.chemeurj.org
945
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