Protein assembly along a supramolecular wire

Article abstract of DOI:10.1039/c0cc02084b

Discotic molecules self-assemble into supramolecular wires that act as platforms for directed protein assembly via appended biotin functionalities.

Full text of DOI:10.1039/c0cc02084b

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Protein assembly along a supramolecular wirewz
Marion K. Mu
¨
ller,a Katja Petkaub and Luc Brunsveld*ab
Received 25th June 2010, Accepted 3rd August 2010
DOI: 10.1039/c0cc02084b
Discotic molecules self-assemble into supramolecular wires that
act as platforms for directed protein assembly via appended
biotin functionalities.
There is great interest in self-assembled nanostructures of
proteins not only because protein assembly and aggregation
play major roles in biological processes,1,2 also because,
protein higher-order aggregates have found strong attention
as novel materials for (bio)nanotechnology applications.3,4
Frequently, these protein assemblies have been obtained by
using proteins or protein elements as building blocks.3–5 The
utilization of synthetic structuring elements provides novel
opportunities to direct and tune protein assembly characteristics
such as stability and shape, either by furnishing the proteins
with synthetic elements6–10 or with ordered synthetic frame-
works that induce and control aggregation.11,12 Supramolecular
polymers have emerged as attractive architectures in nano-
science in the fields of chemistry and biology,13,14 and allow
access to additional larger protein assembly platforms.15
Novel dynamic supramolecular platforms are especially attractive
because their reversible nature will allow reversible adaptivity
in the protein assembly.14
Scheme 1 Discotic monomers decorated with ethylene glycols (1)
self-assemble into inert supramolecular wires, devoid of interactions
with proteins. Discotic monomer provided with biotin handles (3)
assemble in columnar assemblies that induce peripheral protein
assembly. Efficient supramolecular protein assembly is shown by
energy transfer from the supramolecular platform to the proteins
and in between the proteins.
Here we show how an auto-fluorescent, columnar supra-
molecular polymer can be used as self-assembling platform for
protein assembly (Scheme 1). Discotic monomers reversibly
assemble into a columnar polymer at low concentrations in
water (1).16 By providing this self-assembled molecular wire
with ligands (3) we now can utilize this system to assemble
proteins. This is illustrated using biotin as a ligand that
can attract either streptavidin or a biotin-antibody to the
supramolecular platform. The supramolecular scaffold allows
proteins to interact by inducing proximity along the wire.
Discotic 1 (Scheme 2) self-assembles into highly fluorescent
columnar structures at low concentrations in water.16 The
decoration of these supramolecular polymers with ethylene
glycol chains ensures good water-solubility and reduces
unspecific interactions with biological matter.17 Based on
discotic 1, first its analogue 2 with three peripheral amine
functionalities was synthesized using a convergent multistep
approach (see ESIz). The peripheral amine groups were
envisioned to enable rapid final stage modification of the
discotic with ligands of interest. The introduction of the amine
a Chemical Genomics Centre of the Max Planck Society,
Otto-Hahn Straße 15, 44227 Dortmund, Germany
Scheme 2 Chemical structures of discotics 1–3.
b Laboratory of Chemical Biology, Department of Biomedical
Engineering, Technische Universiteit Eindhoven, Den Dolech 2,
5612AZ, Eindhoven, the Netherlands. E-mail: l.brunsveld@tue.nl;
Fax: +31 402478367; Tel: +31 402473737
groups was based on selective mono-alkylation of gallic acid
derivative 10 with a tosylated hexaethylene glycol furnished
with a Boc protected amine. After hydrolysis of the gallic ester
and acid chloride formation, the obtained molecule was
coupled with 3,30-diamino-2,20-bipyridine. Subsequent reaction
with benzene-1,3,5-tricarbonyl chloride led to a Boc protected
w This article is part of the ‘Emerging Investigators’ themed issue for
ChemComm.
z Electronic supplementary information (ESI) available: Synthesis
protocols and spectroscopy protocols. See DOI: 10.1039/c0cc02084b
c
310 Chem. Commun., 2011, 47, 310–312
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discotic, which was deprotected to yield discotic 2. Subsequently
in the final step, 2 was functionalized with biotin by threefold
coupling with NHS-activated biotin in CH2Cl2 under ambient
conditions to yield biotinylated 3. The threefold functionalization
of the discotic proceeds rapidly in molecularly dissolved state,
as non-assembled monomer, in CH2Cl2.
Subsequently, the protein assembly of fluorescently labelled
anti-biotin antibody (AB) and streptavidin (SA) proteins on
supramolecular wires of 1 or of 3 was investigated. First, the
assembly of Cy3s labelled streptavidin (Cy3-SA) and Cy3s
labelled anti-biotin antibody (Cy3-AB) to biotinylated 3
aggregated in water was investigated using Forster resonance
¨
energy transfer (FRET). The dye Cy3s was selected as its
absorbance spectrum overlaps with the emission spectrum of
the discotic molecules 1 and 3. Protein assembly on the
supramolecular wire is expected to induce close proximity of
the Cy3s acceptor fluorophore and the discotic donor
fluorophore. This would lead to a decrease in donor signal
(emmax = 524 nm) with a simultaneous increase in acceptor
signal (emmax = 570 nm). Non-biotinylated 1 was used as a
control to study the interaction between the proteins and the
discotics without any specific binding partners. Mixtures of
supramolecular wires of 116 and the biotin binding proteins
showed only the emission of the discotic donor (Fig. 1, black
Fig. 2 Normalized emission of the discotic donor (3, 1 mM) at 524 nm
plotted against the concentration of Cy3s labelled protein (Cy3-SA
(’) or Cy3-AB (2)). Inset: Fluorescence spectra of the Cy3-AB
titration series. Spectra were recorded by using an excitation wave-
length of 340 nm, and measured in phosphate buffer (pH 7.3).
curve). Indeed, addition of Cy3-SA and of Cy3-AB to supra-
molecular wires formed by 3 resulted in a strong decrease in
donor emission and the appearance of Cy3s acceptor
emission (Fig. 1, red curve). This shows that the proteins are
in close proximity to the columnar structure. This result
shows that both Cy3-labelled streptavidin and Cy3-labelled
anti-biotin antibody are capable of binding to the supramolecular
wires of 3, and that this reaction is selectively induced by the
appending biotins on the supramolecular scaffold. Also the
results show a stronger energy transfer for the Cy3-SA than
the Cy3-AB.
To investigate the protein assembly and the energy transfer
process for both proteins in more detail, titration studies were
performed (for titration curves see Fig. S1 and S2 in ESIz).
Different concentrations (from 0.3 nM to 1.5 mM) of either
Cy3-SA or Cy3-AB were added to a 1 mM solution of 3. Upon
addition of increasing amounts of Cy3-labelled protein, the
fluorescence of the discotic donor decreased up to a complete
quenching of donor fluorescence (Fig. 2). The titration curves
clearly show that a higher concentration of Cy3-AB is required
for efficient energy transfer than of Cy3-SA. For Cy3-AB,
about half of the donor fluorescence is quenched after the
addition of around 0.2 equivalents of Cy3-AB (0.2 mM in
Fig. 2). For Cy3-SA only 0.02 equivalents suffice to quench
half of the donor fluorescence (0.02 mM in Fig. 2). This implies
that at this concentration the fluorescence of approximately
25 discotics in one supramolecular wire is quenched by one
Cy3-SA acceptor fluorophore (donor : acceptor ratio is 50 : 1
and half of donor is quenched). Importantly, this result thus
confirms16 the supramolecular assembly of the discotics in
long molecular wires.
The differences in the FRET effects observed for the two
types of proteins could result from several factors. Firstly, the
antibody has a lower binding affinity for biotin (1.7 ꢀ 10ꢁ9 M)18
in comparison to streptavidin (4 ꢀ 10ꢁ14 M).19 Secondly, the
number of biotin binding sites differs between the antibody (2)
and streptavidin (4). Thirdly, the antibody (B150 kDa) is
three times as large as streptavidin (B50 kDa). This final point
can lead on the one hand to an increased distance between the
Fig.
1
(a) Fluorescence spectra of Cy3s labelled streptavidin
(0.02 mM) incubated with 1 (black) and 3 (red) (both at 1 mM). (b)
Fluorescence spectra of Cy3s labelled anti biotin antibody (0.02 mM)
with 1 (black) and 3 (red) (both at 1 mM). Spectra were recorded by
using an excitation wavelength of 340 nm, and measured in phosphate
buffer (pH 7.3).
c
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In conclusion, supramolecular polymeric wires based on
discotic molecules provide attractive architectures for protein
assembly. The supramolecular materials provide access to
novel framework shapes, such as the columnar wires shown
here, on which proteins can be made to aggregate and interact.
On the supramolecular wire the proteins are brought to
assemble via a specific interaction with biotin ligands displayed
on the periphery. The approach shown here could also be
used to assemble differently labelled proteins and make them
interact as observed via inter-protein FRET.
The supramolecular approach is especially attractive as this
now opens the possibility to attract different types of proteins
to the scaffold simultaneously by utilizing mixtures of
dynamically assembling discotics decorated with a variety of
protein ligands. The supramolecular nature of the scaffold will
allow for easy access to different assembly compositions due to
the exchange of discotics within the wires,14,17 providing this
system with unique properties to tune protein assembly and as
functional material for energy transfer applications in chemical
and materials sciences. Additionally, this supramolecular
system might show potential as functional material at the
single-molecule level.
Fig. 3 Fluorescence spectra of a 1 : 1 mixture of TexasRed and Alexa
Fluor 633 labelled streptavidins (each 0.1 mM) incubated with 1 (black)
and 3 (red) (both at 1 mM). Spectra were recorded by using an
excitation wavelength of 584 nm, and measured in phosphate buffer
(pH 7.3).
dye and the discotic scaffold, and on the other hand to a steric
crowding by the large antibody assembling on the supra-
molecular wire. Both effects would result in a lower FRET
efficiency. The titration results thus show that the protein
efficiently assembles on the supramolecular wire and that the
protein characteristics direct the assembly process.
The research was supported via a Sofja Kovalevskaja
Award of the Alexander von Humboldt Foundation and the
BMBF to LB.
Notes and references
In order to demonstrate the ability of the supramolecular
wire to act as a scaffold capable of inducing protein inter-
actions, two different streptavidins labelled with a FRET pair
(Alexa Fluor 633s and Texas Reds) were assembled on the
supramolecular assemblies formed by 3. The biotin–streptavidin
interaction should mediate the assembly of the streptavidins
on the supramolecular wires and as such facilitate approximation
of the FRET pair. This would lead to a decrease in TexasReds
emission (emmax = 612 nm) and an increase in Alexa Fluor
633s emission (emmax = 647 nm). Alexa Fluor 633-labelled
streptavidin (A633-SA) and TexasRed-labelled streptavidin
(TR-SA) were pre-incubated in a 1 to 1 ratio at a final
concentration of 0.1 mM of each protein. Fluorescence spectra
(excitation = 584 nm) were measured after the addition of 3,
or of 1 as control at 1 mM. The emission spectrum of the
FRET pair in the presence of assemblies formed by 1 (Fig. 3,
black line) is similar to that without discotic (Fig. S3, ESIz).
However in the presence of supramolecular assemblies of 3,
the donor signal of SA-TR almost disappears and the acceptor
fluorescence of SA-AF633 significantly increases (Fig. 3, red
curve). Even though the proteins are present at significantly
lower concentration than the discotic (0.1 vs. 1 mM), an almost
complete quenching of the SA-TR fluorescence is induced.
This effect can only result from the recognition and assembly
of the different streptavidins on the same supramolecular
platform. This protein assembly induces the proximity of
two fluorescently labelled proteins and enables non-radiative
energy transfer from SA-TR to SA-AF633.
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