Dynamic and bio-orthogonal protein assembly along a supramolecular polymer

Article abstract of DOI:10.1039/c3sc50891a

Dynamic protein assembly along supramolecular columnar polymers has been achieved through the site-specific covalent attachment of different SNAP-tag fusion proteins to self-assembled benzylguanine-decorated discotics. The self-assembly of monovalent discotics into supramolecular polymers creates a multivalent, bio-orthogonal and self-regulating framework for protein assembly. The intrinsic reversibility of supramolecular interactions results in reorganization and exchange of building blocks allowing for dynamic intermixing of protein-functionalized discotics between different self-assembled polymers, leading to self-optimization of protein arrangement and distance as evidenced by efficient energy transfer between fluorescent proteins.

Full text of DOI:10.1039/c3sc50891a

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Dynamic and bio-orthogonal protein assembly along a
supramolecular polymer†
Cite this: Chem. Sci., 2013, 4, 2886
Katja Petkau-Milroy,‡a Dana A. Uhlenheuer,‡a A. J. H. Spiering,b
b
a
*
Jef A. J. M. Vekemans and Luc Brunsveld
Dynamic protein assembly along supramolecular columnar polymers has been achieved through the site-
specific covalent attachment of different SNAP-tag fusion proteins to self-assembled benzylguanine-
decorated discotics. The self-assembly of monovalent discotics into supramolecular polymers creates a
multivalent, bio-orthogonal and self-regulating framework for protein assembly. The intrinsic
reversibility of supramolecular interactions results in reorganization and exchange of building blocks
allowing for dynamic intermixing of protein-functionalized discotics between different self-assembled
polymers, leading to self-optimization of protein arrangement and distance as evidenced by efficient
energy transfer between fluorescent proteins.
Received 3rd April 2013
Accepted 26th April 2013
DOI: 10.1039/c3sc50891a
www.rsc.org/chemicalscience
Due to their responsive nature, supramolecular assemblies
offer a promising synthetic platform for dynamic chemistry,19–21
Introduction
bridging the gap to dynamic biological systems.22 Ease of
assembly, modularity, tunability and the potential to incorpo-
rate multiple copies of different active units are further advan-
tages of self-assembled systems ranging from micellar-like
architectures to completely synthetic nanostructures.23–25
The assembly of proteins in cells and on cell membranes is
essential for diverse range of fundamental biological
a
processes including gene transcription, signal transduction and
the regulation of metabolic pathways.1–3 The underlying design
principles of these natural protein assemblies such as well-
dened spatial organization and shape, induced proximity, and
multivalency have inspired the design of synthetic scaffolds for
protein assembly based on small molecules,4–7 dendrimers,8–10
and natural and synthetic polymers,11–17 which exemplify many
of these benecial features with applications in diagnostics and
nanotechnology. Another key element of natural protein
assemblies is their reversibility which serves as a self-regulatory
mechanism, for example during gene transcription, cell motility
and cell division.18 The dynamic assembly and disassembly of
the gene transcription machinery or of actin laments renders
the cellular processes susceptible to changes. Synthetic systems
which possess an inherently dynamic framework for protein
assembly will provide a point of entry to synthetic biological
architectures with dynamic properties and functions that
mimic their biologically inspired counterparts.12
Recently, we have reported
a class of auto-uorescent
C3-symmetrical amphiphilic discotics which instantly assemble
into supramolecular columnar stacks in water.26 These discotics
have been functionalized with different bio-active small mole-
cules, such as sugars or biotin.27,28 Studies with trivalent func-
tionalized discotics and multivalent proteins such as
streptavidin and concanavalin A showed the binding of several
proteins to these self-assembled discotics. The multivalent
nature of these protein–ligand interactions used by us and
others,5–7,15,28 however, limits full molecular control over the
number of proteins appended and hampers investigations into
the dynamics of the supramolecular protein assembly.
Herein we present the site-specic, bio-orthogonal, and
covalent conjugation of single proteins to an intrinsically
monovalent discotic molecule functionalized with one O6-ben-
zylguanine moiety (Fig. 1a). Separate conjugation of two types of
uorescent proteins (yellow and cyan uorescent) allows the
self-assembling discotic scaffold to form supramolecular
architectures that can act as a multivalent and dynamic protein-
assembly platform (Fig. 1b). The dynamic nature of the supra-
molecular polymer allows the uorescent proteins to come
together leading to efficient energy transfer, but also to
dynamically adjust their localization upon building block
exchange and density tuning, resulting in optimized and
adaptable energy transfer (Fig. 1c).
aLaboratory of Chemical Biology and Institute for Complex Molecular Systems,
Department of Biomedical Engineering, Eindhoven University of Technology, Den
Dolech 2, 5612 AZ Eindhoven, The Netherlands. E-mail: l.brunsveld@tue.nl; Fax:
+31 40 2478367
bLaboratory of Macromolecular and Organic Chemistry, Department of Chemistry and
Chemical Engineering, Eindhoven University of Technology, Den Dolech 2, 5612 AZ
Eindhoven, The Netherlands
† Electronic supplementary information (ESI) available: Materials, methods,
detailed
experimental
procedures,
compound
and
self-assembly
characterization, and supplementary gures. See DOI: 10.1039/c3sc50891a
‡ These authors contributed equally.
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Fig. 1 (a) Structure of the O-benzylguanine (BG) discotic 2 carrying a single moiety for conjugation to SNAP-tag fusion proteins. In water these discotics self-assemble
to form auto-fluorescent columnar stacks, displaying moieties for conjugation at their periphery. The photographs show 10 mM solutions of 2 in CH2Cl2 (top) and in
water (bottom) excited with UV light (lex ¼ 350 nm). (b) Site-selective covalent functionalization of the supramolecular polymer with a SNAP-tag fused to cyan and
¨
yellow fluorescent protein (CFP and YFP). (c) Dynamic intermixing of supramolecular protein assemblies results in efficient Forster resonance energy transfer (FRET)
between CFP and YFP. Intercalation of inert-discotic 3 allows tuning of the distance between the fluorescent proteins.
through conversion of acid 4 into the corresponding dichloride
and subsequent amidation with 5 (Scheme 1). The saponica-
tion of the resulting ester 6 was performed using LiOH in water
Results and discussion
Design and synthesis
Covalent, monovalent and site-specic protein conjugation was
sought aer to study the dynamics of supramolecular protein
assembly. Covalent attachment circumvents protein dissocia-
tion and facilitates the characterization of protein-conjugates.
Monovalent and site-specic attachment ensures reproduc-
ibility and a well-dened stoichiometric composition. There-
to prevent hydrolysis of the aromatic amides. A mild chlorina-
tion agent, 1-chloro-N,N,2-trimethylpropenylamine,29 was used
to convert the resulting acid into an acid chloride without
affecting the secondary amide bonds of the molecule. Amida-
tion with 7 bearing a single Boc-protected amine and subse-
quent deprotection afforded the desired desymmetrized
monoamine discotic 1 in high yield. O6-(4-glutarylamidomethyl)-
benzylguanine30 was coupled to this amine-functionalized dis-
cotic 1 in dimethylacetamide using HBTU and DIPEA and
the excess of O6-(4-glutarylamidomethyl)benzylguanine was
removed by size exclusion chromatography to give pure O-ben-
zylguanine-discotic (BG-discotic) 2 in 90% yield.
fore,
a discotic supramolecular building block mono-
functionalized with O6-benzylguanine (2), a reactive function-
ality for bio-orthogonal protein conjugation, was designed,
offering a non-sterically hindered general platform for the
covalent attachment of diverse proteins. This intrinsically
monovalent building block should, only upon self-assembly,
generate a multivalent polymer displaying several proteins in
mutual proximity, but with dynamic adaptability.
Selective mono-functionalization with O6-benzylguanine
required the synthesis of a C2-symmetric discotic molecule
featuring a single amine at one of its nine peripheral oligo-
(ethylene oxide) chains. Desymmetrization was achieved
Functionalization of discotics with uorescent proteins
The specic reaction of O6-benzylguanine with the SNAP-tag,31,32
a genetically encoded protein tag which can be fused to the
protein of interest, provides an entry for site-selective covalent
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Scheme 1 Synthesis of benzylguanine-discotic 2 and structure of the inert-discotic 3. Reagents and conditions: (a) (COCl)2, DMF, CH2Cl2, 0 ꢀC, 1 h, quant., (b) 5, NEt3,
CH2Cl2, RT, 18 h, 52%, (c) (i) LiOH, H2O, 85 ꢀC, 16 h, (ii) aq. oxalic acid, 85%, (d) 1-chloro-N,N,2-trimethylpropenylamine, RT, 2 h, quant., (e) 7, NEt3, CH2Cl2, RT, 18 h, 77%,
(f) TFA, CH2Cl2, RT, 1 h, quant. and (g) O6-(4-glutarylamidomethyl)benzylguanine, HBTU, DIPEA, DMA, RT, 4 h, 90%.
attachment of proteins to discotic 2. This self-labelling tag
The covalent and single protein conjugation to the discotics
offers entry to highly selective and single protein labelling and, was further conrmed by SDS-PAGE and LC-MS. The SDS gel of
in contrast to widely applied bioconjugation techniques such as a ligation mixture of CFP-SNAP with 2, for example, shows a new
thiol–maleimide chemistry, enables labelling in complex band at a higher molecular weight, corresponding to the protein
mixtures, for example for future cellular work.33 Both, yellow covalently linked to 2 (Fig. 2c). Only this band features uo-
and cyan uorescent proteins were therefore expressed and rescence when excited at 350 nm, resulting from the conjugated
puried as SNAP-tag fusion proteins (YFP-SNAP and CFP-SNAP, discotic. No change in protein size and uorescence was
respectively).34 The proteins were conjugated to the self- observed upon incubation with the inert discotic 3 (Fig. 2c). LC-
assembled BG-discotic 2 in phosphate buffer at micromolar MS analysis of the reaction products conrmed the correct
concentrations (see ESI†). The auto-uorescence of the discotics corresponding masses of the discotic functionalized proteins
in the self-assembled state (lexc ¼ 340 nm, lem ¼ 510 nm, (Fig. 2d and S5, ESI†).
dashed line in Fig. 2a) makes these supramolecular polymers
ideal FRET donors for the YFP protein (lexc ¼ 515 nm) and
Supramolecular assembly of multiple proteins
should allow on-line monitoring of the functionalization of 2 via
uorescence
spectroscopy.
Fluorescence
spectroscopy The self-assembling multivalency25,36 of these intrinsically
measurements revealed a time-dependent increase of the YFP monovalent discotics bodes well for the display of different
emission at 527 nm during the protein conjugation reaction proteins along the same supramolecular polymer. The FRET-
upon excitation of the discotics (Fig. 2a). Incubation of YFP- pair proteins YFP- and CFP-SNAP were therefore simulta-
SNAP with inert-discotic 3, not featuring a reactive O6-benzyl- neously ligated to a solution of 2. For this, a 1 : 1 mixture of
guanine handle, did not result in FRET (Fig. 2b). These results YFP-SNAP and CFP-SNAP was added to the self-assembled BG-
prove that the observed energy transfer in case of the BG-dis- discotic 2 and in a control experiment to the self-assembled
cotic 2 with YFP-SNAP is specically due to the covalent protein inert-discotic 3. Only when CFP is in close proximity to YFP the
attachment. Attachment of CFP-SNAP, not being an efficient excited-state energy of CFP is transferred to YFP, resulting in an
FRET acceptor for the discotic, did not lead to changes in the increase in YFP uorescence intensity and a decrease in CFP
self-assembly induced emission spectra of 2. This indicates that uorescence intensity. Only the supramolecular polymer
the protein functionalization of the discotics does not nega- decorated with the reactive O6-benzylguanine handles featured
tively affect their self-assembly behaviour (see Fig. S9, ESI†). The energy transfer from CFP to YFP, resulting in an effective FRET
supramolecular polymer thus ably displays the proteins at its ratio of 0.68 (Fig. 3). The control mixture with the inert
periphery, while efficiently enabling interplay of the chromo- supramolecular polymers featured only CFP uorescence
phores of the polymer with that of the appended proteins.
(Fig. 3, black line). These results show that the intrinsically
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Dynamic intermixing
The modular nature of these supramolecular polymers should
enable the generation of heterovalent supramolecular polymers
via post-assembly intermixing of differently functionalized
discotic building blocks (for concept see Fig. 1c).37 The
exchange of protein-conjugated discotics between different
supramolecular polymers would rearrange the two different
proteins (CFP and YFP), leading to mutual proximity. Thus
dynamic intermixing experiments can easily be followed in real
time using FRET experiments. Supramolecular protein poly-
mers decorated with either YFP or CFP were rst prepared by
separate attachment of the individual proteins to the self-
assembled BG-discotic 2. Subsequently these solutions were
mixed and the appearance of FRET was followed over time
(Fig. 4). A strong time-dependent increase in FRET is observed
aer mixing the two supramolecular protein assemblies.38,39
This evidences that the discotics (monomer ¼ 3.5 kDa) deco-
rated with large proteins (50 kDa) exchange between the
supramolecular polymers. As a control the same protein
mixtures were prepared using inert-discotic 3 and this mixture
did not feature any increase in latent FRET. This also proves the
absence of unspecic protein aggregation overtime as well as
the requirement of covalent attachment of proteins to the dis-
cotic monomers for an increase in energy transfer upon
dynamic intermixing. The monomer exchange is rapid; aer 10
min the half-maximal increase in FRET ratio is reached. The
data is tted best with a double exponential t (Fig. S13, ESI†)
indicating the presence of both a fast and a slower exchange
process, the mechanism behind this is still under investigation.
Fig. 2 (a and b) Emission spectra of a mixture of YFP-SNAP (1 mM) with either
BG-discotic 2 (3 mM, (a)) or inert-discotic 3 (3 mM, (b)) at t ¼ 0 (black) and t ¼ 24 h
(red) measured at 20 ꢀC. The dashed line shows the emission of the discotic before
the addition of protein; lexc ¼ 340 nm. (c) SDS-PAGE of CFP-SNAP (1), and
mixtures of CFP-SNAP with either BG-discotic 2 (2) or inert-discotic 3 (3) after
incubation for 3 h at 37 ꢀC. Left: UV illumination, right: Coomassie blue staining.
(d) ESI-MS spectra and deconvoluted mass of CFP-discotic conjugate (top) and of
CFP-SNAP (bottom).35
Tuning of protein density
The supramolecular nature of the polymers, in principle allows
inserting additional monomers into the main-chain. Therefore,
Fig. 3 Emission spectra (lexc ¼ 410 nm) of 1 : 1 mixtures of YFP-SNAP (1 mM) and
CFP-SNAP (1 mM) incubated for 3 h at 37 ꢀC with either the BG-discotic 2 (20 mM;
red trace) or inert-discotic 3 (20 mM; black trace).
Fig. 4 FRET ratios (lexc ¼ 410 nm) of just mixed samples of 2 separately ligated
to YFP-SNAP and CFP-SNAP (blue) and of just mixed control samples of 3 with
YFP-SNAP and CFP-SNAP (black). Final concentration of discotics is 20 mM and of
proteins 1 mM each. The intermixing of the two protein-functionalized supra-
molecular polymers leads to a heterovalent supramolecular protein polymer
containing both CFP and YFP and results in an increase in FRET ratio over time.
Arrow indicates addition of 20 mM of 3. For corresponding fluorescence spectra
and the non-corrected FRET-ratios see ESI.†
monovalent BG-discotics 2, upon self-assembly, offer a multi-
valent scaffold for protein attachment and assembly, leading to
mutual proximity of different proteins along the supramolec-
ular polymer.
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inert-discotic 3 was added to the mixed supramolecular poly-
mer assemblies of YFP and CFP (Fig. 4, arrow). When inter-
mixing with the already present supramolecular protein
assemblies, the inert-discotics should act as spacers leading to
an increased distance between the uorescent proteins (for
concept see Fig. 1c). Indeed, a rapid decrease in energy transfer
was observed upon addition of 3. Additionally, the distance
between the proteins and with it the decrease in FRET ratio
could be tuned via the addition of different concentrations of 3
(see Fig. S16, ESI†). These results show that in contrast to the
typically applied strategy for the generation of heterovalent
Acknowledgements
The research leading to these results has received funding from
the Ministry of Education, Culture and Science (Gravity
program 024.001.035), from ERC grant 204554 – SupraChemBio
and CTMM – 03O-201 Mammoth.
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