A chaperonin as protein nanoreactor for atom-transfer radical polymerization

Article abstract of DOI:10.1002/anie.201306798

The group II chaperonin thermosome (THS) from the archaea Thermoplasma acidophilum is reported as nanoreactor for atom-transfer radical polymerization (ATRP). A copper catalyst was entrapped into the THS to confine the polymerization into this protein cage. THS possesses pores that are wide enough to release polymers into solution. The nanoreactor favorably influenced the polymerization of Nisopropyl acrylamide and poly(ethylene glycol)methylether acrylate. Narrowly dispersed polymers with polydispersity indices (PDIs) down to 1.06 were obtained in the protein nanoreactor, while control reactions with a globular protein- catalyst conjugate only yielded polymers with PDIs above 1.84.

Full text of DOI:10.1002/anie.201306798

Angewandte
Chemie
DOI: 10.1002/anie.201306798
Nanoreactor
A Chaperonin as Protein Nanoreactor for Atom-Transfer Radical
Polymerization**
Kasper Renggli, Martin G. Nussbaumer, Raphael Urbani, Thomas Pfohl, and Nico Bruns*
Abstract: The group II chaperonin thermosome (THS) from
the archaea Thermoplasma acidophilum is reported as nano-
reactor for atom-transfer radical polymerization (ATRP). A
copper catalyst was entrapped into the THS to confine the
polymerization into this protein cage. THS possesses pores that
are wide enough to release polymers into solution. The
nanoreactor favorably influenced the polymerization of N-
isopropyl acrylamide and poly(ethylene glycol)methylether
acrylate. Narrowly dispersed polymers with polydispersity
indices (PDIs) down to 1.06 were obtained in the protein
nanoreactor, while control reactions with a globular protein–
catalyst conjugate only yielded polymers with PDIs above 1.84.
level in order to gain insight into reaction mechanisms and
catalysis.[8]
Several nanovessels have been investigated as potential
nanoreactors, including swollen domains in polymer net-
works,[6,9] lipid and polymer vesicles,[10] and hollow protein
complexes, so-called protein cages.[1a–e,11] They have in
common that they enclose catalysts, most often enzymes,
into a cavity surrounded by a shell or interface. This boundary
layer is either permeable for substrates and products, or it
encompasses pores that allow for the exchange of matter
between the inside and the outside of the nanoreactor. Thus,
the activity and the substrate selectivity of the enclosed
catalyst do not only depend on the intrinsic properties of the
catalyst, but also on the tailored properties of the nano-
reactor.[1e] Nature provides intriguing nanovessels with
defined pores in the form of protein cages, the best known
being ferritin and viral capsids.[1a–e] Although some protein
cages act as gated nanoreactors in living cells, for example, the
microbial microcompartments,[12] implementations in non-
natural settings with nonnative catalytic species are still
a challenge. Successful examples are a peroxidase enclosed in
the cowpea chlorotic mottle virus[8a] or alcohol dehydrogen-
ase encapsulated into the capsid of bacteriophage P22.[13]
Here, we introduce the thermosome (THS), a group II
chaperonin from the archaea Thermoplasma acidophilum,[17]
as a nanoreactor for polymerization reactions. THS is
a hexadecameric protein complex that consists of eight
a and eight b subunits. In its closed conformation it is
a spherical protein cage about 16 nm in diameter that encloses
two cavities of approximately 130 nm3 each (Scheme 1a).[14] It
provides a folding chamber to refold denatured proteins and
can cycle between open and closed states by the consumption
of ATP.[17] An advantage of THS compared to other nano-
reactors is that the chaperonin, when in its open conforma-
tion, possesses pores that are large enough to allow macro-
molecules to enter and leave the cavities (Scheme 1b).[14,17,18]
Thus, it should be an ideal nanoreactor to synthesize polymers
in its interior with the possibility to release the formed
macromolecules into solution.
A
reactor confines a chemical reaction into a defined
volume. On the macroscopic scale, this is important to control
and modulate the reaction through parameters such as
temperature, stirring, and feeding rates of substrates. If,
however, a reaction is confined in a vessel with dimensions on
the nanoscale, that is, into a yocto liter (= 10À24 L) volume in
a nanoreactor, the course of a chemical reaction can be
influenced in unique ways.[1] Reaction rates can be enhanced
by bringing substrate and catalyst in close proximity,[2] side
reactions can be suppressed by limiting the number of
reactive species that encounter each other,[3] the reactor can
act as a template that defines the size of the formed
products,[4] and the nanoreactor can provide a cavity to
influence single-chain folding of synthetic polymers.[5] Fur-
thermore, reactions can be carried out in environments where
the reaction would normally not occur, such as biocatalysis in
organic solvents[6] or in living cells.[7] Nanoreactors also offer
the possibility to investigate reactions on the single-molecule
[*] Prof. Dr. N. Bruns
Adolphe Merkle Institute, University of Fribourg
Rte de l’Ancienne Papeterie CP209, 1723 Marly (Switzerland)
E-mail: nico.bruns@unifr.ch
K. Renggli, M. G. Nussbaumer, R. Urbani, Prof. Dr. T. Pfohl,
Prof. Dr. N. Bruns
In the field of polymer synthesis, atom-transfer radical
polymerization (ATRP) has proven to be one of the most
versatile and successful controlled/living radical polymeri-
zation techniques, because it tolerates the presence of
numerous functional groups, biomacromolecules, and reac-
tion media.[19] Polymers synthesized by ATRP have been
used, for example, as building blocks for nanostructures,[20] to
form protein–polymer conjugates,[21] and in drug-delivery
systems.[22] Although ATRP in aqueous media has been
demonstrated,[19,21c,23] working in pure water still presents
a challenge since the polymerization can be too fast and side
Department of Chemistry, University of Basel
Klingelbergstrasse 80, 4056 Basel (Switzerland)
[**] This research was supported by a Marie Curie Intra European
Fellowship and a Marie Curie European Reintegration Grant, by the
Holcim Stiftung Wissen, by the Swiss National Science Foundation,
and by the NCCR Nanoscale Science. We thank W. Meier (Depart-
ment of Chemistry) for his support, M. Zehringer (Kantonales
Laboratorium Basel-Stadt) and J. Jourdan (Department of Physics)
for the NAA, V. Olivieri (Zentrum fꢀr Mikroskopie) for the TEM
images, and T. Eaton for proofreading the manuscript.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201306798.
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b subunit was completely modified with a 25-fold excess (with
respect to thiol groups) of 3-maleimido-6-hydraziniumpyri-
dine hydrochloride (MHPH) as determined by UV/Vis
spectroscopy (see the Supporting Information). After purifi-
cation, the modified THS was reacted with 1, a derivative of
the
ligand
N,N,N’,N’-tetraethyldiethylene
triamine
(TEDETA) featuring an aromatic aldehyde moiety
(Scheme 1c). The reaction yields a stable bisaryl hydrazone
bond between the protein and the ligand.[27] The formation of
this linker can be monitored by UV/Vis spectroscopy. The
spectrum of the conjugate in Figure 1a shows the typical
absorption band of proteins at 280 nm as well as the band of
the bisaryl hydrazone link with a maximum at 354 nm. The
deconvolution of this spectrum allows calculating the average
number of bisaryl hydrazone bonds and gave a ratio of ligands
per THS of 3.9 Æ 0.1. The attached ligand was used to complex
CuII ions, followed by purification of the modified THS (THS-
LxCu) by size-exclusion spin centrifugation. The linker can
only have formed inside the THS because of the location of
the engineered cysteine residues. Therefore, the TEDETA–
Cu complex must be entrapped in the cavity of the chaper-
onin. This was confirmed by small-angle X-ray scattering
(SAXS) measurements. THS-LxCu showed an increased
Scheme 1. Structure of THS and strategy to entrap catalysts into the
protein cage. a) Representation of THS in its closed conformation.[14]
b) Cryo-electron microscopy (cryo-EM) density map of a thermosome
in its open conformation (Data from Methanococcus maripaludis,[15]
since no structural data of the THS in this conformation is available in
the databases. This structure is in good agreement with the cyro-EM
data of the THS.[16]). c) Coupling strategy to covalently bind a TEDETA-
derived ligand (1) into the cavities of THS. This ligand was used to
complex CuII, thereby resulting in a protein–catalyst conjugate, that is,
a nanoreactor for ATRP.
reactions tend to play a more important role than in other
reaction media.[19b,24] Confining the reaction space within the
small volume of a nanoreactor should increase the degree of
control over the polymerization and increase the degree of
functionalization at the chain ends of the products.[25]
A strategy to sequester ATRP into a protein nanoreactor
is to conjugate initiators to protein cages.[26] However, this
grafting-from approach does not allow for a continuous
production of polymers since only one polymer chain per
protein-bound initiator is formed and the chain is covalently
attached to the protein. In order to use THS as a nanoreactor
for ATRP, we therefore chose to entrap an ATRP catalyst
into the THS and start chain growth from soluble initiators
that diffuse into the THS. Thus, polymer chains that form in
the protein cage are able to leave the cavities and make room
for further chains to grow.
An organometallic catalyst was conjugated into the
cavities of the THS by using bisaryl hydrazone linker
chemistry (Scheme 1c), similar to our reported method to
entrap fluorescent proteins into THS.[18] A THS mutant that
displays one free cysteine residue on the inside surface of each
Figure 1. Characterization of THS-LxCu. a) The UV/Vis spectrum shows
absorption bands of the protein and of the bisaryl hydrazone that links
^
the catalyst to the protein. b) SAXS data of THS-LxCu ( ) in compar-
*
ison to nonmodified THS ( ) shows a higher contrast induced by the
electron-dense copper that is bound to the ligand in the THS.
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contrast compared to the nonmodified THS (Figure 1b), thus
indicating that the former is a nanoobject with higher electron
density than the THS alone, that is, the engineered THS
contained copper ions. The best fit of the scattering data gave
a diameter of 17.2 nm for THS-LxCu, which is in agreement
with the diameter of the protein in the apo state.[16] Therefore,
it can be concluded that the electron-rich copper ions reside in
the inner cavities of the THS. To elucidate whether the THS
alone, that is, without a TEDETA ligand, also binds copper,
nonmodified THS was incubated in a CuBr2 solution and
purified as described above. The comparison of SAXS data of
this sample with data of THS that had not been exposed to
CuII shows no difference in shape (Figure S1 in the Supporting
Information). This indicates that copper is not complexed by
the THS itself.
The protein cage was analyzed for its structural integrity
by sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS PAGE) and by native PAGE (Figure S2a and b). Both
gels show the same bands for THS, independent of their
modification with the ATRP catalyst. Moreover, transmission
electron microscopy (TEM) images of the protein–catalyst
conjugate show spherical objects with a diameter of approx-
imately 16 nm, which are typical for fully assembled THS
(Figure S2c).[16] Thus, the overall structure of THS was
unaffected by the encapsulated ATRP catalyst.
Figure 2. Characterization of pNiPAAm and pPEGA synthesized with
Polymerizations were conducted by adding THS-LxCu to
an aqueous buffered solution (100 mm sodium acetate,
150 mm NaCl, 80 mm MgCl2, pH 5.2) of the water-soluble
initiator 2-hydroxyethyl-2-bromobutyrate (HEBIB) and the
monomer N-isopropylacrylamide (NiPAAm) in a molar ratio
of [monomer]/[initiator]/[THS-LxCu] 67:1:1.5 ꢀ 10À5 under
argon atmosphere (reaction scheme see Figure S3). An
excess of ascorbic acid (ratio [THS-LxCu]/[ascorbic acid]
1:2.7 ꢀ 105) was added to reduce CuII in situ to its catalytically
active form THS-LxCuI and to regenerate CuII that could
accumulate during the polymerization, according to the
activators regenerated by electron-transfer (ARGET)
ATRP method.[19a] The polymerization was stopped after
20 h by exposure to air and addition of non-deoxygenated
buffer solution. Polymerizations were performed using the
THS nanoreactor in its apo state, that is, in the absence of
ATP. In this state both lids of the protein cage are open.[15,17]
Thus, any macromolecules that are synthesized within the
THS can leave the cavities after polymerization. Poly(N-
isopropyl acrylamide) (pNiPAAm) was separated from the
protein cage by size-exclusion spin centrifugation, leaving the
THS in the supernatant while the polymer could be collected
from the flow-through. Matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry (MALDI-ToF
MS) of the latter showed a series of signals spaced D(m/z)
113.2, with a number average molecular weight (Mn) of
1500 gmolÀ1 and a polydispersity index (PDI) of 1.11 (Fig-
ure 2a). Since the molecular weight of the monomer is
113.16 gmolÀ1, these data show that the reaction yielded
pNiPAAm. The low PDI indicates that the polymerization
proceeded with a good degree of control. A detailed analysis
of the peaks is shown in Figure S4.
THS-LxCu and BSA-LxCu. a) MALDI-ToF MS data of pNiPAAm synthe-
sized with THS-LxCu in aqueous buffer. b) GPC trace of pNiPAAm
synthesized by BSA-LxCu in aqueous buffer. c) Comparison of GPC
traces from pPEGA synthesized with THS-LxCu (black) or with BSA-
LxCu (gray) in a water/THF (7:3 v/v) mixture.
catalyst conjugated to the outside of the globular protein
bovine serum albumin (BSA-LxCu; characterization see
Figure S5). ARGET ATRP with BSA-LxCu yielded
pNiPAAm with an apparent molecular weight Mn of
42600 gmolÀ1 (calibrated against poly(methyl methacrylate))
and a PDI of 1.94 as determined by gel permeation
chromatography (GPC; Figure 2b). By comparing the two
different catalytic systems (THS as protein nanoreactor
versus the globular protein BSA) we draw the conclusion
that the synthesized polymers are substantially shorter and
have a lower PDI if produced in the nanoreactor. A reason for
the broad molecular-weight distribution of polymers synthe-
sized with BSA-LxCu may be the very low concentration of
catalyst in the reaction. This can be neglected in the THS
since the growing polymer chain stays in close proximity to
the catalyst inside the nanoreactor. Therefore, the synthesis of
pNiPAAm, which is intrinsically difficult to control in
aqueous solution,[24] benefits from the confined reaction
space. The lower Mn of polymers synthesized in the nano-
reactor could be due to diffusion limitations of the monomer
into the protein cage, due to space constrictions of the
growing chain within the cavities, or due to a less probable
reactivation of a dormant chain once it has diffused out of the
nanoreactor.
The scope of THS as nanoreactor for polymerizations was
further explored by conducting the experiments with a differ-
ent monomer. Poly(ethylene glycol) methyl ether acrylate
(PEGA) with a number average molecular weight of
To set our findings into perspective, polymerizations were
conducted under the same conditions, but with the ATRP
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480 gmolÀ1 was polymerized with THS-LxCu under the same
ARGET ATRP conditions used for pNiPAAm, yielding
A set of control reactions was carried out to ensure that
only the Cu–TEDETA complex in the THS bears copper that
is catalytically active in ATRP. Nonmodified THS was
incubated with CuBr2 solution. Then, size-exclusion spin
centrifugation was used to remove the unbound copper ions.
The resulting THS was used to polymerize NiPAAm and
PEGA under the conditions described above. The reactions
pPEGA with
a poly(ethylene oxide)-apparent Mn of
14700 gmolÀ1 and a PDI of 1.95, as analyzed by GPC
(reaction scheme in Figure S3). In comparison, the BSA-
conjugated catalyst produced pPEGA with apparent Mn of
119400 gmolÀ1 and a PDI of 3.12. In addition, the polymer-
ization of PEGA with THS-LxCu was run in the presence of
ATP, which can cause the main pores of THS to cycle between
open and closed states.[17] The reaction resulted in pPEGA
with an apparent Mn of 17500 gmolÀ1 and a PDI of 2.50. The
addition of ATP had no beneficial effect on the polymeri-
zation, possibly because hydrolysis of ATP by THS is too slow
at room temperature, that is, at the reaction temperature of
the polymerizations.[28]
1
did not yield polymer products as checked by H NMR and
GPC. These experiments, in combination with the SAXS data
discussed above (Figure S1), indicate that copper ions are not
complexed by the protein. Further, we assessed whether the
bisaryl hydrazone linker could complex copper ions in an
ATRP-active form. For this purpose an analogue of the BSA-
Lx was produced that did bear the linker, but no TEDETA
ligand (Figure S6). The modified protein BSA-4FB was mixed
with CuBr2 solution. After size-exclusion spin centrifugation
it was used as catalyst for ARGET ATRP of NiPAAm with
the same reaction conditions as described above. The
polymerization with BSA-4FB did not yield polymer as
Addition of organic cosolvents to aqueous ATRP can
result in better performance of a given catalyst, since side
reactions, for example, disproportionation of the ATRP
activator, loss of the halide ligand by the ATRP deactivator
as well as fast chain propagation are suppressed, thus resulting
in better-controlled polymerizations.[19b] Therefore, PEGA
was polymerized in a 7:3 (v/v) mixture of water and THF, by
using THS-LxCu as catalyst (see Figure S3). The product of
this experiment resulted in an apparent Mn of 1400 gmolÀ1
and a PDI of 1.06 (Figure 2c). BSA-LxCu yielded pPEGA
with an apparent Mn of 14900 gmolÀ1 and a PDI of 1.84. The
PDI and the average molecular weight of pPEGA synthesized
in the presence of organic cosolvent are lower than the ones
obtained without cosolvents. In addition, these experiments
confirm that reactions carried out in nanoreactors yield
polymers with a narrower distribution of molecular weight.
For the ATRP to be confined into the THS, the protein
cage has to be stable during the polymerization. The most
harsh reaction condition was the one with THF as cosolvent.
It was therefore assessed whether or not the THS retained its
structure in a typical PEGA polymerization in the water/THF
mixture. After the reaction, SDS and native PAGE (Fig-
ure 3a,b) show the distinct bands of the two subunits and of
the fully assembled THS. Moreover, TEM (Figure 3c)
indicates that the protein cage remained intact. Thus, the
protein cage was stable in this reaction.
1
checked by H NMR and GPC. This control reaction with
BSA-4FB indicates that, even though copper ions might be
complexed by the linker, only TEDETA in combination with
copper ions results in complexes that are able to catalyze
ATRP. This underscores the necessity for conjugating a suit-
able Cu-binding ligand into the THS to obtain an ATRP
catalyst inside of the protein cage.
In conclusion, we introduced the chaperonin THS as
a nanoreactor and conducted ATRP in the protein cage by
binding an ATRP catalyst to the inner cavities of the protein.
To our knowledge, this is the first reported protein–catalyst
conjugate for ATRP. THS is particularly appealing for this
application because it has pores that are large enough to allow
macromolecules to enter and leave the cavities. The synthe-
sized polymers are therefore released into the surrounding
solution without having to disassemble the protein cage into
its subunits. We showed that the confined space within the
nanoreactor results in polymers with narrow molecular
weight distributions. Such a positive effect on the polymer-
ization is most probably due to the proximity of the ATRP
reagents and is consistent with simulations of other nano-
reactor systems.[25b] Protein nanoreactors could influence
polymerizations in some unique ways, for example, by
imparting selectivity for certain monomers, or by modulating
the rate of the polymerization through triggers that close or
open pores in the protein wall. As chaperonins are the folding
chambers of nature, they could beneficially affect the folding
of (block)copolymers when sequestered single polymer
chains are synthesized within their cavities. In addition, the
use of protein nanoreactors could enable mechanistic studies
of ATRP on a single-molecule level.
Received: August 3, 2013
Revised: September 18, 2013
Published online: December 20, 2013
Figure 3. Characterization of THS-LxCu after the polymerization in
a water/THF (7:3 v/v) mixture. a) SDS gel electrophoresis, b) native
gel electrophoresis, and c) TEM images of THS-LxCu recovered after
polymerization (THSrec-LxCu). These data allow the conclusion that
THS remained stable during polymerization and retained its structure.
Keywords: chaperone proteins · controlled/
living radical polymerization · nanoreactor · polymerization ·
thermosome
.
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