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Communication
Towards Artificial Mitochondrion: Mimicking Oxidative
Phosphorylation in Polymer and Hybrid Membranes
Lado Otrin, Nika Maruši#, Claudia Bednarz, Tanja R. VidakovicKoch, Ingo Lieberwirth, Katharina Landfester, and Kai Sundmacher
Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b03093 • Publication Date (Web): 25 Oct 2017
Downloaded from http://pubs.acs.org on October 26, 2017
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Towards Artificial Mitochondrion: Mimicking
Oxidative Phosphorylation in Polymer and Hybrid
Membranes
Lado Otrin,† Nika Marušič,† Claudia Bednarz,† Tanja Vidaković-Koch,†,* Ingo Lieberwirth,‡
Katharina Landfester,‡ and Kai Sundmacher†,§
†
Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106
Magdeburg, Germany
‡
Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany
§
Otto von Guericke University, Universitaetsplatz 2, 39106 Magdeburg, Germany
* Corresponding author: vidakovic@mpi-magdeburg.mpg.de, +49 391 67 54630
Abstract: For energy supply to biomimetic constructs, a complex chemical energy-driven ATPgenerating artificial system was built. The system was assembled with bottom-up detergentmediated reconstitution of an ATP synthase and a terminal oxidase into two types of novel
nanocontainers, built from either graft copolymer membranes or from hybrid graft
copolymer/lipid membranes. The versatility and biocompatibility of the proposed nanocontainers
was demonstrated through convenient system assembly and through high retained activity of
both membrane-embedded enzymes. In future, the nanocontainers might be used as a platform
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for the functional reconstitution of other complex membrane proteins and could considerably
expedite the design of nanoreactors, biosensors and artificial organelles.
Keywords: Nanoreactors, nanocontainers, energy regeneration, artificial organelle, hybrid
materials, bottom-up.
Bottom-up synthetic biology emerged from the ambitious goals of few pioneering groups
(Luisi, Yomo, Szostak and others) to understand the principles of life and its origin and the
aspiration to imitate it through reverse engineering. Since its inception, mainly natural
components of living cells including enzymes, fatty acids and genetic material were isolated,
explored and orchestrated to mimic or reimagine life processes, and to design new, applicationdriven ones. To this end the genetic code was synthesized and manipulated into genetic circuits.13
Meanwhile, proteins were refined through directed evolution, produced by cell-free expression,
and arranged into versatile metabolic cascades.4-6 Finally, under the guidance of engineering
principles, natural building blocks were integrated towards the construction of protocell
models.7-9 An important awareness, which materialized during the assembly of such life-like
systems, was the importance of designing efficient schemes for continuous energy regeneration,
necessary to drive life and life-mimicking processes alike.
In addition to reiterating life, bottom-up synthetic biology offers a unique opportunity of
combining man-made and biological parts for creating augmented biological systems, which do
not exist in nature but resemble certain features of living systems.10,11 Supplementation of natural
components with artificial ones is often necessary to circumvent particular shortcomings of
biological materials. The combination of synthetic polymers and biological components, such as
integral membrane proteins (IMPs), appears especially appealing since polymers provide
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excellent mechanical stability, tunable environment and robustness, while the proteins introduce
their intrinsic functionalities.12 Some utilization of similar artificial systems, comprised of
functionalized polymer containers, was seen over the last decade in medicine and pharmacology
as drug delivery systems13-15 and contrasting agents16-18 as well as in biotechnology as
nanoreactors19-21 and biosensors22,23. However, for several applications (drug delivery,
construction of artificial organelles etc.), functionalization of polymer containers with more
complex, environment-sensitive membrane proteins, is a necessity.
At this point, predominantly very rigid proteopolymersomes formation methods seem to vastly
limit the set of IMPs suitable for functional reconstitution to the few robust, most stable, simple
model IMPs, capable of membrane integration through self-assembly, such as ompF, Tsx, AQP0
and AqpZ.24-27 Meanwhile, more and more IMPs had been successfully reconstituted into
liposomes each year and more complex systems, comprised of two or more co-reconstituted
IMPs had been formed.28-30 Reconstitutions of IMPs into liposomes are in the majority of cases
mediated by the use of detergents. A possibility to apply similar detergent-based reconstitution
methods, optimized for high performance of the reconstituted membrane protein, to suitable
polymer or hybrid membranes, would considerably expedite the formation of new versatile
nanoreactors, biosensors and complex artificial systems.
A first step towards the integration of more complex IMPs into polymersomes was made by
Choi and Montemagno by the integration of bacteriorhodopsin in the form of purple membranes
along with ATP synthase into polymersomes, using the triblock copolymer PEtOz-PDMSPEtOz.31 The light-driven synthesis of ATP was successfully demonstrated in this multiprotein
polymersome system and it was attributed to the biocompatibility of the tested polymer.
However, no evidence presented in this study suggested that the observed functionality of ATP
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synthase was not instead facilitated by the large amount of lipids introduced to the polymersomes
in the form of purple membranes.
A recent example, that clearly demonstrated how the functionality of the environment-sensitive
membrane protein was sacrificed for higher overall stability and durability of the artificial
system, was the one of cytochrome bo3 quinol oxidase (bo3 oxidase) reconstituted in diblock
copolymer PBd-PEO containers as well as in more complex hybrid PBd-PEO/POPC
containers.32 While the bo3 oxidase retained most of its activity in hybrid containers featuring
high lipid content (50-75 %), a staggering 70 % reduction of activity was observed in hybrid
containers at low lipid content (25 %). Furthermore, bo3 oxidase reconstituted in pure
polymersomes showed no significant activity, exposing the poor biocompatibility of the PBdPEO membranes. Nevertheless, this study revealed the high relevance of hybrid lipid/polymer
containers to the emerging field of synthetic biology, for which the stability and durability of the
artificial systems are as important as the high retained functionality of all membrane-embedded
components of the system.
In this Letter we take a step away from the commonly used block copolymer-based
nanocontainers
and
introduce
versatile,
stable,
biocompatible
and
immunostealth33
nanocontainers based on the graft copolymer poly(dimethylsiloxane)-graft-poly(ethylene oxide)
(PDMS-g-PEO). In aqueous solutions this polymer self-assembles into stable vesicular
structures34 with a reported membrane core thickness of 5 nm.35 Due to the polymer membrane
thickness similar to the one of a typical lipid bilayer and its high fluidity of 371 cP, (e.g.
viscosity of E. coli membrane extract is 320 cP and of E. coli cells is 1160 cP at 23 °C)36 PDMSg-PEO is a promising substitute for classical lipid membranes. Furthermore, PDMS-g-PEO can
be mixed with lipids to form hybrid lipid/polymer membranes with the composition-dependent,
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predictable homogeneous or heterogeneous (phase-separated) distribution of both components in
the membranes.37 Conveniently, polymer or hybrid vesicles with controlled mean diameters can
easily be obtained by successive extrusion (SUVs, LUVs), while larger vesicles (GUVs), suitable
for vesicle characterization, can be produced using the electroformation procedure.
An artificial system, capable of chemical energy-driven ATP production (schematically
represented in Fig. 1C) was established through bottom-up detergent-mediated integration of
purified transmembrane proteins, the bo3 oxidase and the F1FO ATP synthase, into the proposed
novel nanocontainers. In parallel, its natural counterpart, optimized for performance (bo3
oxidase/ATP synthase proteoliposomes) was adapted as a benchmark for the evaluation of
biocompatibility of our nanocontainers. The bo3 oxidase, a chemical energy-driven proton pump,
was used to establish a proton gradient, sufficiently high to drive the synthesis of ATP. The
prerequisites for the successful ATP synthesis in this system were: 1) a functionally
reconstituted, active bo3 oxidase with the quinol binding site well accessible to ubiquinol-1 (Q1,
the electron shuttle), 2) a tight, proton-impermeable membrane and 3) a membrane capable of
facilitating the rotational motion of ATP synthase as well as the proton translocation by both
enzymes. Thickness and fluidity of the artificial membranes in particular are expected to play a
crucial role in functional reconstitution of the described system. Finally, the integration of the
described system was established through a detergent-mediated reconstitution. The versatility of
the polymer and hybrid nanocontainers in regard to system´s assembly was demonstrated with
detergents, commonly used for the reconstitution of IMPs into liposomes, namely sodium cholate
(SC), sodium deoxycholate (SDC) and octyl β-D-glucopyranoside (OG).
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Figure 1. (A) Fluorescence microscopy images of homogeneous hybrid microcontainer
comprised of 70 mol% PDMS-g-PEO and 30 mol% of soy PC. The distribution of lipid (left) and
polymer (middle) is homogeneous in well-mixed (right) hybrid membranes. (B) Intensity-based
size distribution of hybrid (top) and polymer (bottom) nanocontainers after the extrusion, with
their corresponding polydispersity indexes indicated in the bottom-right corner of each plot. (C)
Schematic representation of the ATP generating system. Measured variables, used for the
determination of the activity of both enzymes in lipid (L), polymer (P) or hybrid (H)
nanocontainers are marked with red squares.
For the purpose of characterization of polymer and lipid distribution in hybrid membranes, first
micron-sized hybrid containers were prepared with the electroformation procedure from, for the
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first time, natural lipid extract soy phosphatidylcholine (soy PC) and PDMS-g-PEO, in various
polymer-to-lipid ratios. To enable observation of both components of the nanocontainers, the
fluorescein-labelled polymer probe was synthesized (PDMS-g-PEO-fluo) and the distribution of
both components in the artificial membranes was analyzed by use of fluorescence microscopy.
Homogeneous, well-mixed membranes were observed at and above 70 mol% of polymer in the
membranes (Fig. 1A), while phase-separated membranes were observed at lower polymer
concentrations (SI Fig. 1). Homogeneous hybrid microcontainers were stable for at least several
weeks. Similar results were reported for polymer/synthetic POPC hybrid microcontainers.37 In
the following experiments, homogeneous hybrid nanocontainers intended for later system
integration were prepared with a polymer-to-lipid mol% ratio of 75:25, featuring the highest
stability in the case of hybrid microcontainers.
For the reconstitution of both membrane proteins, nano-sized polymer containers as well as
homogeneous hybrid nanocontainers were produced with the extrusion technique. Morphology
of the artificial nanocontainers was determined by cryo-EM imaging. Nanocontainers exhibited
hollow vesicular structure with the average membrane thickness of 5 nm (Figure 2), as reported
previously35,37. Intensity-based size distribution and polydispersity of nanocontainers were
tracked by dynamic light scattering (DLS). Both types of nanocontainers were uniform in size
(Fig. 1B) with Z-averages (105 nm for hybrids and 102 nm for polymersomes) being close to the
diameter of pores of the extrusion filter (100 nm). A low polydispersity index, very similar to the
one of liposomes, was obtained for both types of nanocontainers.
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Figure 2. Cryo-EM micrographs of hybrid nanocontainers (A) and polymer nanocontainers (B).
Insets: Both types of the artificial nanocontainers with membrane-embedded enzymes.
In the next step, the activity of the bo3 oxidase reconstituted in hybrid and polymer
nanocontainers (reconstitution described in the SI) was monitored. The changes in oxygen
concentration during a turnover of the bo3 oxidase were detected with the Clark-type electrode
(Fig. 3A). The bo3 oxidase was reconstituted with all tested detergents and the activities were
compared to the activities of the enzyme in liposomes (SI Fig. 2 and SI Fig. 3).
Figure 3. (A) Changes in oxygen concentration due to the activity of bo3 oxidase in hybrid
nanocontainers. At t ≈ 170 s mark, the reaction was initiated by ubiquinone-1 (Q1). The trace
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shown is for the hybrid nanocontainers with membrane-embedded enzyme reconstituted with 0.2
% SDC. The oxygen consumption rate was determined from the slope indicated as “v0”. (B) The
comparison of highest obtained oxygen consumption rates for the natural and two artificial
systems with the corresponding values expressed in nmol of O2 ml-1 min-1.
With the measured oxygen consumption rates as high as 35.8 nmol ml-1min-1 (hybrids) and
34.9 nmol ml-1min-1 (polymersomes), we were able to prove the successful functional integration
of bo3 oxidase into both types of nanocontainers. Compared to proteoliposomes (Fig. 3B) only a
minimal loss of activity was observed. Having in mind previous reports32 on bo3 oxidase
reconstitution in polymersomes and hybrid vesicles, where the enzyme was completely
deactivated upon integration in polymersomes and retained only 30 % of its original activity in
hybrid vesicles, the activity of bo3 oxidase found in the present study, in both hybrid and
polymer vesicles, is truly remarkably high. We believe that high activity of the enzyme can be
attributed mainly to smaller thickness (5 nm) and higher fluidity of the proposed artificial
membranes when compared to much thicker (> 10 nm) and highly viscous block copolymer
membranes.32 Quinol binding site of bo3 oxidase, located in the transmembrane region of the
enzyme, is more accessible to Q1 in thinner membranes, which more closely reflect natural
membrane thickness. Furthermore, high fluidity of the proposed artificial membranes enables
lateral diffusion of Q1 towards the quinol binding site of bo3 oxidase, upon the addition of
mentioned cofactor to the artificial energy regeneration system.
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Figure 4. (A) Changes in luminescence generated by luciferin/luciferase system as a result of
respiration-driven ATP synthesis. Proton-pumping by the bo3 oxidase was initiated at t = 200 s
mark by the addition of ubiquinone-1 (Q1). The slope normalized against a known amount of
ATP represents the ATP synthesis rate. The trace shown is for the energy regeneration system
reconstituted in hybrid nanocontainers with 0.05 % OG. (B) The comparison of highest obtained
ATP synthesis rates for natural, hybrid and polymer-based artificial system with the
corresponding values, expressed in molecules of ATP/ATP synthase/s.
The complete artificial system was assembled with octyl glucoside, the detergent which was
shown to reconstitute highly active bo3 oxidase. Octyl glucoside was added to the preformed
hybrid nanocontainers or was introduced to polymer nanocontainers during their formation (for
further details please see SI) at or below the membrane saturation point (Rsat)38. Detergentsaturated artificial membranes were supplemented with both membrane proteins and their
incorporation was induced by the dilution of protein-containing micelles below the CMC of the
stabilizing detergents and by removal of octyl glucoside from the artificial membranes by BioBeads adsorbent resin. Bio-Beads were added in excess (6.6 times their binding capacity for OG;
reconstitution mixture contained maximum amount of 0.8 % or 0.8 mg OG while the binding
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capacity of 45 mg of the Beads was 5.3 mg OG39) to assure total detergent removal. The
efficiency of the detergent removal was confirmed by mass spectrometry. It was found that 99.95
% of reconstitution-mediating detergent was removed from lipid nanocontainers, whilst over
99.99 % of the reconstitution-mediating detergent was removed from both hybrid and polymer
nanocontainers (SI Table 1). While we assumed that all added enzymes were integrated into
nanocontainers, the reconstitution efficiency is very likely lower than a 100 %, which might have
led to the underestimation of the activity of ATP synthase. Furthermore, while the ATP synthase
is known to incorporate with the exclusive orientation of more than 97 % of the F1 moieties
(“head”) facing outwards40, the orientation of bo3 oxidase in the membranes was shown to be
random41. Therefore, one can assume that only the nanocontainers with more than 50 % of
correctly oriented bo3 oxidase (facing inwards) contributed to the net proton influx and the
formation of ATP by ATP synthase. Accordingly, other nanocontainers remained silent, leading
to further underestimation of turnover number of ATP synthase.
Respiration-driven ATP synthesis rates of proteonanocontainers were determined based on
bioluminescence produced by the luciferin/luciferase CLSII reagent42 (Fig. 4A). A comparison
of the highest achieved ATP synthesis rates of the natural, hybrid and polymer-based artificial
systems are depicted in Fig. 4B (for the comparison of ATP synthesis rates of hybrid and
polymer nanocontainers see SI Fig. 4). An overall highest ATP synthesis rate of 11 ATP/ATP
synthase/s was observed in liposomes, reconstituted with 0.6 % SC. Interestingly, the highest
ATP synthesis rates of proteohybrid and polymer nanocontainers of 6.1 ATP/ATP synthase/s and
3.6 ATP/ATP synthase/s, respectively, were observed at very low concentrations of OG (0.05
and 0.1 % OG) for both types of nanocontainers. Due to the relatively high cost of detergents, the
nanocontainers, presented in this Letter might be considered an attractive, cost-efficient
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alternative to liposomes. The artificial energy regeneration system was able to continuously
produce ATP for at least several hours at 6× higher ATP synthesis rate than a similar, lightdriven system31, reconstituted in hybrid nanocontainers. With the sufficient supply of co-factors
to the artificial system, physiological levels (0.5–5 mM) of the produced ATP can be reached,
which is a prerequisite for a large number of biological and biomimetic processes. While we
believe that the co-reconstitution of ATP synthase with another membrane protein into artificial
containers, presented in this study, could certainly be improved further, we conclude that the
achieved high ATP synthesis rates of this complex system are a good indicator of the excellent
biocompatibility of the proposed novel nanocontainers.
In this study we presented a new type of stable, versatile, stealth and fully biocompatible
nanocontainers based on graft copolymer PDMS-g-PEO. The copolymer alone was used for the
formation of polymersomes or was combined with natural lipid extract for the formation of
hybrid lipid/polymer nanocontainers. We demonstrated an excellent uniformity of both types of
nanocontainers, reflected in consistent size, low polydispersity and a good control over the
distribution of both components in hybrid membranes. The presented nanocontainers offer a
versatile platform for the reconstitution of various new and complex membrane proteins. Bo3
oxidase functionalized hybrid nanocontainers could serve as pH-switchable encapsulated
substrate-processing nanoreactors. Additionally, PDMS-g-PEO polymersomes, functionalized
with the desired IMPs, may also show great potential as stealth drug delivery systems. Unlike
commonly used block copolymer-based delivery systems, graft copolymer-based nanocontainers
can readily be dissolved to release the encapsulated substance.
With the bottom-up approach and through the employment of commonly used detergents, we
integrated two purified enzymes into both types of nanocontainers to form complex, respiration-
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driven ATP-generating artificial systems. High activity of both integrated enzymes further
indicated excellent biocompatibility as well as integrity of the artificial membranes. The
presented artificial system, capable of continuous production of ATP, shows great promise in the
exciting, rapidly developing field of synthetic biology, where it could be used to supply energy
to countless natural and synthetic energy-demanding biomimetic constructs.
Supporting Information Available:
Text describing materials and methods, figures SI Fig. 1, SI Fig. 2, SI Fig. 3 and SI Fig. 4, table
SI Table 1. (PDF)
The following files are available free of charge at http://pubs.acs.org.
ACKNOWLEDGMENT
This work is part of the MaxSynBio consortium which is jointly funded by the Federal Ministry
of Education and Research (BMBF) of Germany and the Max Planck Society. The authors are
grateful to Prof. C. von Ballmoos and his team from the University of Bern for the generous gift
of two plasmids used in this study and for invaluable suggestions and discussion. The authors are
also grateful to Ivan Ivanov, Anne Christin Reichelt and Drew Rudman for their assistance
during various stages of manuscript preparation.
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