Cucurbit[8]uril induced heterodimerization of methylviologen and naphthalene functionalized proteins

Article abstract of DOI:10.1039/c1cc11197c

Cucurbit[8]uril is a supramolecular inducer of protein heterodimerization for proteins appended with methylviologen and naphthalene host elements. Two sets of fluorescent protein pairs, which visualize the specific protein assembly process, enabled the interplay of the supramolecular elements with the proteins to be established.

Full text of DOI:10.1039/c1cc11197c

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Chemical Communications
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Volume 47 | Number 24 | 28 June 2011 | Pages 6733–6992
ISSN 1359-7345
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Luc Brunsveld et al.
Cucurbit[8]uril induced heterodimerization of methylviologen and naphthalene functionalized proteins

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Cite this: Chem. Commun., 2011, 47, 6798–6800
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Cucurbit[8]uril induced heterodimerization of methylviologen and
naphthalene functionalized proteinsw
a
b
Dana A. Uhlenheuer,z Jacqui F. Young,z Hoang D. Nguyen,b Marcel Scheepstraa and
Luc Brunsveld*ab
Received 28th February 2011, Accepted 5th April 2011
DOI: 10.1039/c1cc11197c
Cucurbit[8]uril is a supramolecular inducer of protein hetero-
dimerization for proteins appended with methylviologen and
naphthalene host elements. Two sets of fluorescent protein pairs,
which visualize the specific protein assembly process, enabled the
interplay of the supramolecular elements with the proteins to be
established.
Controlled generation or stabilization of protein complexes is of
high interest for applications in for example the biomedical or
bionanotechnology fields. Supramolecular approaches are in
principle very attractive for this since they allow bioorthogonal
control over these interactions,1 thus complementing the
protein engineering and small molecule approaches.2 A number
of supramolecular approaches for the controlled assembly of
proteins have been reported in this respect based on, for
example, host–guest chemistry1 or on the combination of
natural protein assemblies with synthetic polymer chemistry.3
Synthetic supramolecular host–guest systems are very attractive
for the generation of small well-defined protein assemblies
such as homo- and heterodimers. The strong cyclodextrin–
lithocholic acid interaction for example, could be applied for
the controlled supramolecular heterodimerization of proteins in
Scheme 1 Supramolecular induced protein dimerization by formation of
solution and in cells, by appending these supramolecular
elements to the C-terminus of the proteins.4,5 The supra-
molecular host molecule cucurbituril has also found strong
application in the protein assembly and immobilization field.6
Proteins genetically encoded with an N-terminal FGG-motif
can for example be assembled into a homodimer via two-fold
binding of the peptide motif within cucurbit[8]uril (CB[8]).7
CB[8] is not only known to form 1 : 2 complexes with peptide
motifs,8 but also to form stable 1 : 1 : 1 ternary complexes with
an electron deficient supramolecular guest molecule such as
methylviologen (MV) and an appropriately corresponding electron
rich guest such as alkoxynaphthalene (Np).9,10 These two guest
a ternary complex between methoxynaphthol and methylviologen–CB[8].
molecules form a charge transfer complex inside the CB[8]
cavity. Here we demonstrate that this specific ternary supra-
molecular system can be used to induce the selective hetero-
dimerization of two proteins, with CB[8] acting as
a
supramolecular inducer of dimerization (Scheme 1). Further-
more we show that there is a distinct interplay of the supra-
molecular host–guest system with the proteins. Two sets of
fluorescent protein FRET pairs allowed establishing the specific
characteristics of the CB[8]–MV–Np system with respect to
protein assembly and unspecific protein aggregation.
a Laboratory of Chemical Biology, Department of Biomedical
Engineering, Technische Universiteit Eindhoven, Den Dolech 2,
5612 AZ Eindhoven, The Netherlands. E-mail: l.brunsveld@tue.nl;
Fax: +31 40 247 8367; Tel: +31 40 247 3737
We selected methoxynaphthol (Np) and methylviologen
(MV) as the supramolecular guest pair to form a ternary
complex with CB[8]. For the site-selective attachment of these
supramolecular groups to recombinantly expressed proteins
via expressed protein ligation, the elements were to be
provided with a cysteine moiety. The proteins we selected are
two sets of cyan and yellow fluorescent protein (CFP and YFP)
FRET pairs. The two sets of fluorescent proteins differ in their
b Chemical Genomics Centre of the Max Planck Society,
Otto-Hahn Str 15, 44227 Dortmund, Germany
w Electronic supplementary information (ESI) available: Synthetic
protocols, analytical data, fluorescence spectroscopy. See DOI:
10.1039/c1cc11197c
z These authors contributed equally to this work.
c
6798 Chem. Commun., 2011, 47, 6798–6800
This journal is The Royal Society of Chemistry 2011

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Scheme 2 Synthesis of cysteine modified guest elements methylviologen (MV) and naphthalene (NP) and their ligation to two sets of fluorescent
proteins: enhanced CFP and YFP (eCFP, eYFP) and dimerizing CFP and YFP (dCFP, dYFP). (a) MeI in CH3CN, RT, 24 h;
(b) 3-bromopropylamineꢀHBr in CH3CN, reflux, 24 h; (c) BocCysStBu–OSu, DIPEA, CH3CN, 40 1C, 4 h; (d) 30% TFA, CH2Cl2; (e) TCEP,
MeOH/phosphate buffer pH 7.5, RT, 30 min; (f) eYFP–thioester or dYFP–thioester in phosphate buffer pH 7.5, RT, 12 h; (g) NaH, 6-bromo-
hexan-1-ol, 0 1C, DMF, RT; (h) BocCysStBu–OH, EDCꢀHCl, DMAP, DMF; (i) 20% TFA, CH2Cl2; (j) TCEP, MeOH/Tris–HCl buffer pH 8.5,
Triton, RT, 2 h; (k) eCFP–thioester or dCFP–thioester in Tris–HCl pH 8.5, Mesna, RT, 12 h.
intrinsic affinity for dimerization due to hydrophobic
mutations. These were chosen as they allow the supramolecular
assembly process to be followed using fluorescence techniques
that specifically visualize the interaction between the proteins.
This will provide information of the assembly process on the
protein level, in contrast to, for example, optical techniques
that visualize host–guest assembly in the CB[8] cavity.11 This
allows the interplay of the supramolecular elements with the
protein assembly process to be studied and to distinguish
from unspecific protein assembly induced by, for example,
hydrophobic clustering.
expressed and purified analogously as previously described,5
and ligated to the cysteine-modified host molecules. The
YFP–thioester variants were reacted with an excess of MV–Cys
in the presence of thiophenol for 12 hours to give complete
conversion. The naphthalene based host molecule Np–Cys
showed limited solubility in the ligation buffer and ligations
to the CFP variants were therefore performed in the presence of
detergents to enhance the solubility and reaction speed.
The CB[8] controlled protein assembly of the two protein
FRET pairs was studied using fluorescence spectroscopy.
Fluorescence spectra were recorded using an excitation
wavelength of 410 nm which is optimized for selective CFP
excitation. The supramolecular-induced protein heterodimers
were prepared by addition of CB[8] to the specific methyl-
viologen-modified YFP, followed by the addition of the
naphthalene-modified CFP. A complete set of experiments
including all possible reference samples was thus made for
both protein pairs (Fig. S1 and S2, ESIw).
The normal variants of the fluorescent proteins were initially
investigated (Fig. 1). As envisioned in our design, CB[8] indeed
selectively induces the heterodimerization of MV–eYFP with
Np–eCFP. This becomes clearly apparent from the increase in
the YFP emission at 527 nm (Fig. 1, blue line), which indicates
the occurrence of FRET upon the addition of CB[8]. This
results in a YFP/CFP ratio (I(527 nm)/I(475 nm)) of 0.79, in
contrast to a ratio of 0.51 for the non-assembled proteins. This
CB[8]-induced high energy transfer between the proteins is
only observed in the presence of all three supramolecular
components, allowing the formation of the ternary complex.
In all other cases, such as for protein pairs lacking either one
or two of the supramolecular units or in the absence of CB[8],
The viologen based host molecule was synthesized from
commercially available bipyridine (Scheme 2). Methylviologen
was obtained by slow addition of methyliodide to 4,40-bipyridine.
The mono-methylated product was further reacted with
3-bromopropylamine hydrobromide to give an asymmetric
substituted viologen derivative12 which was coupled to a
Boc- and StBu-protected cysteine–succinimide ester in aceto-
nitrile. The cysteine conjugate was purified by preparative
HPLC and subsequently the Boc group was removed with
TFA/DCM to yield MV–Cys. The naphthalene based host
molecule was synthesized from commercially available
6-methoxy-naphthalen-2-ol. First, an alcohol spacer was
attached to the phenolic functionality of the naphthalene using
6-bromo-hexan-1-ol and subsequently the primary alcohol
group was coupled to a protected cysteine derivative. After
purification via preparative HPLC, the resulting compound
was Boc-deprotected to give Np–Cys. Deprotection of the
cysteine StBu disulfide functionalities of MV–Cys and
Np-Cys was performed immediately prior to protein ligation
by treatment with TCEP. The four protein thioesters were
c
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Chem. Commun., 2011, 47, 6798–6800 6799

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with either Np–dCFP or unmodified dCFP feature an increase
of the YFP emission in the absence of CB[8]. Interestingly,
however, in the presence of CB[8] the unspecific protein
assembly induced by the methylviologen is inhibited, as
becomes apparent from the absence of YFP emission
(Fig. 2, red line). The CB[8] thus shields the methylviologen
from making unspecific interactions with hydrophobic protein
surfaces. As a result, in the MV–dYFP–Np–dCFP mixture
with CB[8] (Fig. 2, blue line), a specific CB[8] mediated energy
transfer is observed, resulting from the supramolecular protein
dimer. These results show that the ternary system of CB[8]
with MV and Np can also be successfully used for the
formation of selective protein heterodimers of more hydro-
phobic proteins. In these cases, the presence of CB[8] as a host
molecule is required to prevent MV induced unspecific
dimerization with hydrophobic protein surfaces.
Fig. 1 Fluorescence spectra of normal enhanced fluorescent protein
pairs, normalized at the CFP emission maximum and FRET ratio
I(527 nm)/I(475 nm). Protein concentration
concentration 10 mM.
1 mM each, CB[8]
In conclusion, we have shown that CB[8] constitutes an
attractive supramolecular inducer of protein heterodimeriza-
tion using MV and Np as protein appended host elements. The
model system of fluorescent protein pairs allows specific
visualization of the protein dimerization event. This in turn
allows for detection of potential unspecific interplay of the
supramolecular elements with the proteins and provides
molecular insights not attainable using experiments solely
based on the observation of the charge-transfer complex. This
novel system for controlled protein heteroassembly now
allows, for example, application in the area of bionano-
technology for the controlled immobilization of proteins on
surfaces, or for specific targeting of N-terminal tryptophan
motifs in proteins.16
no FRET was observed. Also the formation of protein homo-
dimers upon addition of CB[8] could be excluded via homo-
FRET studies (see ESIw). This demonstrates that the chosen
supramolecular host–guest system is specific and selectively
induces supramolecular heteroassembly of the two proteins.
The other pair of proteins under study, MV–dYFP and
Np–dCFP, carries specific point mutations13 which increase
their intrinsic affinity for dimerization (both homo- and
heterodimerization) due to increased hydrophobic inter-
actions. In general, protein–protein interactions are frequently
mediated to a significant extent by hydrophobic and charged
regions on their surfaces,14 allowing for supramolecular
recognition.15 As such, knowledge about the possible interplay
of the supramolecular elements with the protein surface is
highly important. The weakly dimerizing fluorescent model
proteins MV–dYFP and Np–dCFP are an ideal system to
study this.
Funded by ERC grant 204554—SupraChemBio.
Notes and references
1 D. A. Uhlenheuer, K. Petkau and L. Brunsveld, Chem. Soc. Rev.,
2010, 39, 2817–2826.
2 A. Fegan, B. White, J. C. T. Carlson and C. R. Wagner, Chem.
Rev., 2010, 110, 3315–3336.
3 I. C. Reynhout, J. J. L. M. Cornelissen and R. J. M. Nolte, Acc.
Chem. Res., 2009, 42, 681–692.
4 L. Zhang, Y. Wu and L. Brunsveld, Angew. Chem., Int. Ed., 2007,
46, 1798–1802.
Control experiments performed on the Np–dCFP with
unfunctionalized dYFP and CB[8] indeed showed that these
supramolecular elements do not lead to unspecific binding or
protein interactions (see ESIw). In contrast, experiments with
MV–dYFP showed that the methylviologen can lead to
unspecific protein assembly of the weakly dimerizing proteins
(Fig. 2, green and black lines). Protein mixtures of MV–dYFP
5 D. A. Uhlenheuer, D. Wasserberg, H. Nguyen, L. Zhang, C. Blum,
V. Subramaniam and L. Brunsveld, Chem.–Eur. J., 2009, 15,
8779–8790.
6 See for example: D.-W. Lee, K. M. Park, M. Banerjee, S. H. Ha,
T. Lee, K. Suh, S. Paul, H. Jung, J. Kim, N. Selvapalam, S. H. Ryu
and K. Kim, Nat. Chem., 2010, 3, 154–159.
7 H. D. Nguyen, D. T. Dang, J. L. J. van Dongen and L. Brunsveld,
Angew. Chem., Int. Ed., 2010, 49, 895–898.
8 L. M. Heitmann, A. B. Taylor, P. J. Hart and A. R. Urbach,
J. Am. Chem. Soc., 2006, 128, 12574–12581.
9 Y. H. Ko, E. Kim, I. Hwang and K. Kim, Chem. Commun., 2007,
1305–1315.
10 U. Rauwald, F. Biedermann, S. Deroo, C. V. Robinson and
O. A. Scherman, J. Phys. Chem. B, 2010, 114, 8606–8615.
11 F. Biedermann, U. Rauwald, J. M. Zayed and O. A. Scherman,
Chem. Sci., 2011, 2, 279–286.
12 N. Asakura, T. Hiraishi, T. Kamachi and I. Okura, J. Mol. Catal. A:
Chem., 2001, 174, 1–5.
13 J. L. Vinkenborg, T. H. Evers, S. W. A. Reulen, E. W. Meijer and
M. Merkx, ChemBioChem, 2007, 8, 1119–1121.
14 O. Keskin, A. Gursoy, B. Ma and R. Nussinov, Chem. Rev., 2008,
108, 1225–1244.
15 A. J. Wilson, Chem. Soc. Rev., 2009, 38, 3289–3300.
16 J. J. Reczek, A. A. Kennedy, B. T. Halbert and A. R. Urbach,
J. Am. Chem. Soc., 2009, 131, 2408–2415.
Fig. 2 Fluorescence spectra of dimerizing fluorescent proteins pairs,
normalized at the CFP emission maximum and FRET ratio I(527 nm)/
I(475 nm). Protein concentration 1 mM each, CB[8] concentration 10 mM.
c
6800 Chem. Commun., 2011, 47, 6798–6800
This journal is The Royal Society of Chemistry 2011