Strong supramolecular control over protein self-assembly using a polyamine decorated β-cyclodextrin as synthetic recognition element

Article abstract of DOI:10.1039/c1jm12736e

The supramolecular host molecule heptakis-[6-deoxy-6-(2-aminoethylsulfanyl) ]-β-cyclodextrin provides strong control over protein self-assembly in synthetic supramolecular protein constructs. Mono-functionalization of this modified β-cyclodextrin with a cysteine residue allows for site-selective synthetic conjugation to a protein and formation of a highly stable synthetic protein complex with a lithocholic acid conjugated protein as the interaction partner.

Full text of DOI:10.1039/c1jm12736e

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Volume 21 | Number 47 | 21 December 2011 | Pages 18849–19172
Including articles on the theme of materials chemistry in the emerging field of synthetic biology
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Luc Brunsveld et al.
Strong supramolecular control
over protein self-assembly using a
polyamine decorated β-cyclodextrin as
synthetic recognition element
0959-9428(2011)21:47;1-A

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Strong supramolecular control over protein self-assembly using a polyamine
decorated b-cyclodextrin as synthetic recognition element†‡
Dana A. Uhlenheuer,x Lech-Gustav Milroy,x Pauline Neirynck and Luc Brunsveld*
Received 14th June 2011, Accepted 4th August 2011
DOI: 10.1039/c1jm12736e
The supramolecular host molecule heptakis-[6-deoxy-6-(2-amino-
ethylsulfanyl)]-b-cyclodextrin provides strong control over protein
self-assembly in synthetic supramolecular protein constructs. Mono-
functionalization of this modified b-cyclodextrin with a cysteine
residue allows for site-selective synthetic conjugation to a protein
and formation of a highly stable synthetic protein complex with
a lithocholic acid conjugated protein as the interaction partner.
controlled self-assembly of protein heterodimers in buffered solu-
17
16
18
d
tion and in cells. With a K z 1 mM and given the ease with
which mono-functionalized derivatives of these host–guest molecules
can be prepared, this supramolecular complex is thus well suited to
protein dimerization at micromolar concentrations. However, since
multiple protein-protein interactions occur at sub-micromolar
concentrations, host–guest complexes with higher affinity are
required to enable the controlled formation of supramolecular
protein dimers at even lower concentrations than has thus far been
possible. Recently, Nitz et al. reported that lithocholic acid and
heptakis-[6-deoxy-6-(2-aminoethyl-sulfanyl)]-b-cyclodextrin (2) form
Biomolecules such as proteins interact via reversible supramolecular
interactions, controlling localization and function. Gaining control
over these self-assembly mechanisms is key to understanding and
targeting (dysfunctional) biological processes in the disease state.
Synthetic supramolecular materials chemistry offers a key entry point
to generate completely novel synthetic biological systems with
7
ꢁ1
a host–guest complex with K ¼ 5.5 ꢀ 10 M (K ¼ 0.02 mM) in
a
d
1
9
buffered solution at pH 7.5. Applying this strong synthetic complex
for protein assembly would in principle provide an entry point for
supramolecular control over protein dimerization at concentrations
lower than has been achieved using the ‘naked’ b-cyclodextrin-based
system.
1–7
orthogonal biomimetic functions. In the field of supramolecular
chemical biology, the usage of synthetic supramolecular host–guest
systems is especially attractive in this respect, due to the biochemical
origin of many of these systems and their concomitant bioapplic-
2
0
In a first experiment, 2 was evaluated as an inhibitor of the
supramolecular protein complex formed between a monomeric lith-
ocholic acid-modified monomeric cyan fluorescent protein (mCFP)
and a b-cyclodextrin-modified monomeric yellow fluorescent protein
8
ability. Synthetic host–guest systems can for example be applied to
the controlled and reversible dimerization of proteins, by chemical
appendage of supramolecular elements to proteins in a site-selective
9
manner. Such systems enable the induction or stabilization of
16,17
(mYFP).
Monomeric fluorescent protein variants of this kind are
mutated at the amino acid position 206 – from an alanine to a lysine
21,22
(A206K) – in order to suppress intrinsic protein dimerization.
protein-protein interactions in a manner, which is orthogonal to
The
1
0
existing protein domain-based approaches. Similarly, supramolec-
interaction between mCFP and mYFP can be conveniently followed
by fluorescence spectroscopy, measuring F o€ rster Resonance Energy
23
Transfer (FRET). Thus, the addition of increasing equivalents of 2
ular host molecules can be used to recognize specific protein
1
1–13
elements,
dimerization.
and applied as supramolecular inducers of protein
4,15
Here, we demonstrate the novel use of heptakis-[6-
1
led to the progressive inhibition of the synthetic supramolecular
protein dimer, which could be observed by a decrease in the FRET
signal at 527 nm (Fig. 1). Encouragingly, 2 was found to induce a loss
of FRET signal in a concentration range 10 times lower than that
measured for the normal b-cyclodextrin 1 (ESI, Supporting
Figure 10). Through an enhancement of binding affinity, 2 thus
competes more effectively than 1 with the protein dimer, and provides
an entry for exploration in synthetic protein conjugates.
deoxy-6-(2-aminoethylsulfanyl)]-b-cyclodextrin as a strong chemical
building block for the controlled assembly and disassembly of
synthetic biological architectures.
Previously, we showed how the host–guest complex formed
between lithocholic acid and b-cyclodextrin (1) can be used for the
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
The above reported result prompted us to selectively modify the
mYFP protein with this synthetic supramolecular building block to
strongly induce biological self-assembly. While the syntheses of C7-
symmetric cyclodextrin analogues derived from 2 are already known,
†
Electronic supplementary information (ESI) available: Experimental
20,24,25
details, compound characterization and fluorescene spectroscopy data.
See DOI: 10.1039/c1jm12736e
including a number of impressive applications,
mono-func-
26
tionalized variants have hardly been addressed. Here, we describe
the novel synthesis and purification of a mono-functionalized
heptakis-[6-deoxy-6-(2-aminoethylsulfanyl)]-b-cyclodextrin cysteine
‡ Funded by ERC grant 204554 – SupraChemBio. Ralf Bovee and Joost
van Dongen are thanked for analytical support.
x These authors contributed equally to this work.
This journal is ª The Royal Society of Chemistry 2011
J. Mater. Chem., 2011, 21, 18919–18922 | 18919

1
9,20,25,27
1.
Therefore, 1 was first converted at the C6 position to the
28
bromide in accordance with literature protocol. The obtained per-6-
bromo-b-cyclodextrin was then treated with cysteamine hydrochlo-
2
0
ride under basic conditions to yield pure 2, which was to be
subsequently reacted with N-Boc- and S-StBu-protected cysteine.
The S-StBu protective group is strategically incorporated to enable
in situ deprotection immediately prior to protein ligation. Unlike the
mono-functionalization of b-cyclodextrin, which is a well studied and
2
9
much used reaction sequence, the mono-functionalization of 2 is
now being investigated. We predicted that treating 2 with a suitably
pre-activated cysteine derivative would produce a complex mixture.
and reasoned, due to the excellent water solubility of 2, that the
desired mono-functionalized derivative (3) could potentially be
separated using reversed-phase HPLC. In practice, the protected
cysteine derivative was thus pre-activated as the hydroxysuccinimide
30
ester and added drop-wise to a DMSO solution of 2 in the presence
of excess triethylamine. As expected, formation of mono-substituted
product 3 could be observed by LC/MS, along with unreacted 2 and
the di- and tri-substituted cyclodextrin, presumably as ill-defined
mixtures. Somewhat surprising, however, was the high degree of
separation achieved in this case between 3 and the remaining
components (ESI, Supporting Figure 2), which formed an excellent
basis for further purification. Therefore, using an optimized gradient
on a reversed-phase HPLC system, it was possible to separate 3 from
the other cyclodextrin components present in >95% purity (ESI).
After successful isolation of the mono-functionalized 3, deprotection
of the Boc-group was performed in a quantitative manner using 95%
Fig. 1 (A) Used host–guest molecules, (B) reversal of supramolecular
protein dimer, monomeric fluorescent proteins represented by backbone
folds of cyan (mCFP) and yellow (mYFP) fluorescent proteins, (C)
fluorescence emission spectra showing disappearance of FRET signal due
to increasing concentration of polyamine cyclodextrin 2. Fluorescence
emission spectra of mCFP-mYFP synthetic protein complex (0.5 mM,
2
TFA/H O to yield the pure trifluoroacetate salt 4 for use in our
planned protein ligation studies.
The mono-functional cysteine-cyclodextrin derivative 4 was then
ligated to mYFP, which had previously been successfully ligated to
(CFP)
l
exc
410 nm) before (black) and after addition of 0.5 mM (red) and 1.5
16
the normal b-cyclodextrin (1). We chose the monomeric fluorescent
protein in this case because, for this variant, the affinity of the
supramolecular protein dimer is determined predominantly by the
supramolecular host–guest interaction and not by any intrinsic
mM 2, showing a disappearance of the mYFP emission at around 527 nm.
derivative for attachment to a fluorescent protein via expressed
16,17
protein ligation
16
(Scheme 1). The known hydrochloride salt of 2
binding affinity between the two proteins. Therefore, 4 was first
can be prepared in two steps starting from commercially available
thiol-deprotected using an excess of TCEP in phosphate buffer and
ꢂ
Scheme 1 Synthesis of mono-functionalized cysteamine-b-CD (4) and ligation to the thioester of mYFP: (a) N-bromo-succinimide, PPh , DMF, 75 C,
3
2
8
30
3 3 2
N in DMF, 72 h, rt, 89%; (c) BocCysStBu-OSu, Et N, DMSO, 22 h, rt, 13%; (d) 95% TFA/H O, 3 h, rt,
4
h, 60%; (b) cysteamine hydrochloride, Et
quantitative; (e) phosphate buffer pH 7.5, TCEP, MPAA, rt, overnight, 60%.
1
8920 | J. Mater. Chem., 2011, 21, 18919–18922
This journal is ª The Royal Society of Chemistry 2011

then added directly to the protein thioester. Ligation of the protein
was performed overnight at room temperature using 18 eq. of 4. To
remove the excess of ligand, the buffer was exchanged after ligation to
TrisHCl (pH 8) containing 50 mM NaCl. In the LC/MS spectrum of
the protein ligation product, a small quantity of non-functionalized
protein could be detected. For purification of the desired protein-
conjugate, ion exchange chromatography was applied. It was
reasoned that the ligation of the positive highly-charged cyclodextrin
to the protein would facilitate its sequestration from the starting
protein thioester based on charge differences. Assuming protein
modification with 4 to be equal to the introduction of six unprotected
lysine side chains, a difference in isoelectric point (pI) of 1.1 between
the modified (pI ¼ 6.8) and the unmodified protein (pI ¼ 5.7) was
calculated. A strong anion exchange resin was then selected for
purification, and the protein eluted with increasing concentrations of
NaCl. The first fractions contained pure modified protein (named
and a decrease in mCFP emission at 475 nm. In practice, a very
strong FRET signal was observed for the protein mixture containing
both functionalized proteins: mCLA and mYCDNH (Fig. 2, blue).
2
This strong FRET effect clearly indicated that a supramolecular
protein hetero-dimer was formed by virtue of the host–guest
2
elements. In a control experiment, mYCDNH was mixed with the
non-functionalized mCFP thioester protein. In this case, no increase
in FRET was detected (Fig. 2, black), showing that cyclodextrin 4
itself does not induce unspecific protein assembly. Interestingly, direct
comparison of the two b-cyclodextrin-based supramolecular protein
dimers (mYCD and mYCDNH ) revealed a significantly higher
2
FRET effect for the mYCDNH based complex (Fig. 2, blue), with
2
a mYFP/mCFP ratio (I(527 nm)/I(475 nm)) of 1.18 compared to 0.67
for the mYCD based complex. This finding is in agreement with the
original design of the host molecule 4, which is expected to show
significantly higher binding affinity for lithocholic acid compared
with b-cyclodextrin in the micromolar regime, and possibly facilitate
a better orientation for energy transfer. These results show that the
supramolecular host–guest complex of lithocholic acid and heptakis-
[6-deoxy-6-(2-aminoethylsulfanyl)]-b-cyclodextrin is a highly efficient
synthetic system for the controlled assembly of two proteins.
2
mYCDNH hereafter) which was followed by increasing amounts of
unmodified protein. Analysis via SDS-PAGE and LC/MS showed
that the purification strategy was successful (ESI).
Protein assembly was next investigated using fluorescence spec-
troscopy. To study the applicability of the newly-modified cyclo-
dextrin 4 for supramolecular protein dimerization, the ligated protein
mYCDNH2 was mixed with mCFP ligated to lithocholic acid
In conclusion, we have demonstrated that 2 and novel mono-
functionalized derivative 4 are two attractive synthetic supramolec-
ular elements for the controlled assembly and disassembly of
proteins. A facile synthetic approach to mono-functionalized hepta-
kis-[6-deoxy-6-(2-aminoethylsulfanyl)]-b-cyclodextrin was devised
and the resulting synthetic building block successfully conjugated to
a fluorescent protein. This optimized synthetic host–guest complex
enabled enhanced energy transfer between two proteins at lower
concentrations than had been previously achieved, resulting in
a roughly two-fold increase in mYFP/mCFP ratio at 0.5 mM.
Alternatively, the known b-cyclodextrin derivative 2 is a valuable
inhibitor of synthetic supramolecular protein assembly in its own
right, capable of complete inhibition of supramolecular protein
(mCLA). The efficiency of the mYCDNH –mCLA assembly was
2
compared with a synthetic supramolecular protein dimer based on
the ‘naked’ b-cyclodextrin (mYCD-mCLA). Fluorescence spectra
were recorded using an excitation wavelength of 410 nm which is
optimized for mCFP excitation and performed in phosphate buffer
containing 1 mM TCEP. The TCEP was added to ensure that no
disulfide bridges were formed between the free cysteine residues
1
6
remaining after protein ligation. The two synthetic proteins were
mixed in a 1 : 1 ratio and fluorescence emission spectra were recorded
(Fig. 2). In principle, successful protein heterodimerization results in
FRET, which is visible as an increase in mYFP emission at 527 nm
Fig. 2 Fluorescence emission spectra of 1 : 1 mixtures of 0.5 mM for each protein in phosphate buffer containing 1 mM TCEP; mCFP and mYCDNH
2
(reference, black), mCLA and mYCD (red), mCLA and mYCDNH
2
(blue). Fluorescence spectra were recorded using an excitation wavelength of 410
nm which is optimized for mCFP excitation.
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J. Mater. Chem., 2011, 21, 18919–18922 | 18921

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