Supramolecular Assemblies for Transporting Proteins Across an Immiscible Solvent Interface

Article abstract of DOI:10.1021/jacs.7b13245

Polymeric supramolecular assemblies that can effectively transport proteins across an incompatible solvent interface are described. We show that electrostatics and ligand-protein interactions can be used to selectively transport proteins from an aqueous phase to organic phase. These transported proteins have been shown to maintain their tertiary structure and function. This approach opens up new possibilities for application of supramolecular assemblies in sensing, diagnostics and catalysis.

Full text of DOI:10.1021/jacs.7b13245

Communication
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Supramolecular Assemblies for Transporting Proteins Across an
Immiscible Solvent Interface
Jingjing Gao, Bo Zhao, Meizhe Wang, Mahalia A. C. Serrano, Jiaming Zhuang, Moumita Ray,
Vincent M. Rotello, Richard W. Vachet,* and S. Thayumanavan*
Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, United States
*
S Supporting Information
ABSTRACT: Polymeric supramolecular assemblies that
can effectively transport proteins across an incompatible
solvent interface are described. We show that electrostatics
and ligand−protein interactions can be used to selectively
transport proteins from an aqueous phase to organic
phase. These transported proteins have been shown to
maintain their tertiary structure and function. This
approach opens up new possibilities for application of
supramolecular assemblies in sensing, diagnostics and
catalysis.
Figure 1. Schematic representation of reverse micelle driven protein
transportation.
details). By distributing these polymers in toluene along with
two equivalents of water per carboxylate or quaternary
ammonium moiety, assemblies with a fairly homogeneous
size distribution of 50 nm for P1 and 37 nm for P2 were
observed, as discerned by both dynamic light scattering (DLS)
(Figure 2c) and transmission electron microscopy (TEM)
(Figure 2d,e, S1).
The key premise for the work here is that these self-
assembled structures would bind to complementarily charged
proteins in the aqueous phase, and ferry them across the
interface to the interior of the reverse micelles in toluene. To
test this possibility, porcine liver esterase (plE, MW = 16.8
kDa), a negatively charged enzyme at pH 8.0 (pI = 5.3), was
used as the model protein. Upon equilibrating a plE solution
with cationic P2 (1 mg/mL) reverse micelles, presence of
proteins in both phases was detected using matrix assisted laser
desorption/ionization mass spectrometry (MALDI-MS). Figure
3a,b shows the MS of the aqueous and organic phases,
respectively, before equilibration. After equilibration, we were
gratified to observe a peak corresponding to plE in the organic
phase (Figure 3c), suggesting that plE was successfully
transported into interior of the reverse micelles.
ransporting molecules across incompatible interfaces is a
T
significant challenge, especially for macromolecules. A
striking example of an interfacial barrier is the cellular
membrane, where an organized presentation of hydrophilic
and hydrophobic functional groups provides a formidable
1
barrier for molecular transport. Whereas small ions or
molecules can cross the membrane through ion channels or
passive diffusion, globular proteins with large hydrophilic
2
,3
surfaces offer no easy access. Nonetheless, cells do transport
proteins when necessary for intercellular communication, often
4
,5
using nanoscopic vesicular compartments called exosomes.
Inspired by these cell-derived vesicles, we became interested in
exploring the possibility of transporting proteins into a
nanoscopic compartment across a solvent interface. Indeed,
small molecule surfactants mostly and occasionally polymers
have been explored as a means of loading proteins into reverse
6
−9
micelles.
Loading selectivity with such systems is limited or
nonexistent, even in an electrostatics context, presumably due
10
to the low stability of small molecule based micelles. Though
transporting proteins across interfaces has many implications,
selective transport, while retaining structure and function, could
be transformative in applications such as sensing, delivery, and
diagnostics. Supramolecular assemblies have shown great
To quantify the extent of protein that was encapsulated
within the reverse micelles, we analyzed the organic phase for
proteins using the bicinchoninic acid (BCA) assay. This
analysis showed that 1 mg of polymer is capable of transporting
and binding to 0.05 mg of plE, an equivalent of 5 wt % loading
capacity. This estimate is consistent with the corresponding
SDS-PAGE analysis of the liberated proteins (Figures S6 and
S7). This capacity compares more favorably than that with
11−13
potential in these areas
and supramolecular protein
transport would be an important advance. Here we report a
simple polymeric platform that selectively transports water-
soluble proteins from an aqueous phase to the water-pool of
reverse micelles in an apolar organic phase based on
complementary electrostatic interactions or specific ligand-
protein binding interactions (Figure 1).
14
liposomes. A more compelling analysis is to identify whether
the extracted enzyme remains active in the reverse micelle. To
investigate this possibility, we designed an amphiphilic
For the initial proof-of-concept, we synthesized a polystyr-
ene-based amphiphilic anionic random copolymer P1 (M = 11
n
kDa, Đ = 1.09) (Figure 2a) and a cationic polymer, P2, (Figure
Received: December 14, 2017
2
b) using nitroxide-mediated polymerization (see SI for
©
XXXX American Chemical Society
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DOI: 10.1021/jacs.7b13245
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
Figure 2. Structural features of polymeric reverse micelles. Molecular structure of polymer P1 (M = 11 kDa, Đ = 1.09) (a) and P2 (b) (M = 12
n
n
kDa, Đ = 1.15), (c) DLS profile of P1 and P2 in toluene, TEM of P1 (d) and P2 (e).
Figure 3. MALDI-MS analysis of (a) aqueous phase before equilibration, (b) organic phase before equilibration, and (c) organic phase after
equilibration. (d) Activity of esterase (based on substrate cleavage) inside reverse micelles compared with esterase activity in bulk aqueous phase.
coumarin-based profluorophore S1 as a substrate for plE. The
alkylated ether of this substrate is nonfluorescent. When the
ester bond of S1 is cleaved by the enzyme, the resultant
hemiacetal rapidly degrades to generate umbelliferone, a
fluorescent coumarin molecule (see SI for details).
The substrate itself was quite stable in PBS buffer as well as
after equilibration with toluene solution containing P2. In the
presence of plE, however, a rapid hydrolysis of S1 to generate
the fluorescent umbelliferone was observed (Figure 3d). We
then analyzed the possibility of this reaction in toluene in the
presence of reverse micelles loaded with plE. Interestingly, the
hydrolysis rate was found to be quite similar to that of the free
enzyme, suggesting that the plE activity is maintained inside the
reverse micelles. As another control experiment, we mixed plE
and P2 in aqueous phase and found that the activity of the
enzyme was slightly lower, suggesting that interactions between
P2 and plE have little effect on plE activity.
When considering the pathway by which these polymers
could transport proteins across the interface, we equilibrated
the reverse micelle assemblies of polymers P1 and P2 with
water. UV−visible absorption spectra of both phases indicate
that these polymers fully remain in the apolar phase (Figure
S8). Though these polymers were initially assembled as
micelles in the aqueous phase and equilibrated with toluene
(
Figure S9), the exclusive presence of these polymers in the
aqueous phase shows that these assemblies are kinetically
trapped in the solvent that they are initially assembled. Overall,
these results suggest protein molecules can exchange between
phases, but only remain in the apolar phase if they have
favorable interactions with the reverse micelle interior.
Following these observations, we were interested in exploring
the applicability of this approach to other nonenzymatic
proteins. Green fluorescent protein (GFP) was chosen, not
only because it can be readily monitored using fluorescence, but
also because the fluorescence itself is a good indicator of
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DOI: 10.1021/jacs.7b13245
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Journal of the American Chemical Society
Communication
Figure 4. Emission spectrum of GFP before and after transport, (a) GFP (−7) transport by P2, (b) GFP (−7) transported by P1, (c) GFP (+15)
transported by P1, (d) GFP (+15) transported by P2.
Figure 5. (a) Molecular structure of P3, (b) fluorescence change of aqueous/organic phase using P3 or P4 for bCA transport, (c) molecular structure
of P4, (d) fluorescence change of aqueous/organic phase of P3 to transport bCA, Myb or Lyz, (e) MALDI-MS analysis of protein mixture before and
after transportation.
whether the protein maintains its tertiary structure. Wild-type
GFP (pI 6.2) has a net charge of −7 at pH 7.4, we treated this
GFP (−7) solution with P2 reverse micelle solution in toluene.
We were gratified to find that the emission spectrum of the
organic phase clearly showed the presence of GFP (Figure 4a).
MALDI-MS peak with a m/z of 28 432 Da further confirmed
the presence of GFP in the organic phase (Figure S10).
However, there was no discernible change in the emission
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DOI: 10.1021/jacs.7b13245
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Journal of the American Chemical Society
Communication
intensity of the aqueous phase (Figure 4b) using the anionic
reverse micelle P1, suggesting that this transport is indeed due
to electrostatic complementarity.
control experiments using myoglobin (Mb) and fluorescently
labeled lysozyme (Lyz). Because benzenesulfonamide ligands
have little to no binding affinity to these proteins, we predicted
that Mb and Lyz would remain in the aqueous phase. Indeed,
no discernible fluorescence change was observed in both
aqueous and organic phases for these two proteins, suggesting
that the ligand attached reverse micelles are specific for the
target protein bCA (Figure 5d). These experiments were
initially done separately due to the possible bleeding of
fluorescence emission. Selective transport from a mixture of
these proteins by P3 was tested using MS. We were gratified to
find that only bCA is transported to the organic phase, whereas
Mb and Lyz remained in the aqueous phase as indicated by the
mass spectra before and after equilibration (Figure 5e). These
data strongly support the idea that ligand-attached reverse
micelle systems are specific for target proteins.
To further test this idea, we utilized the so-called
15
supercharged GFP (+15). Indeed, the anionic polymeric
reverse micelle from P1 is able to transport the protein across
the interface, whereas the cationic reverse micelle from P2 does
not affect the protein in the aqueous phase (Figure 4c,d). The
results from these studies show that (i) transport of proteins
across the interface is due to electrostatic complementarity, not
due to spurious differences in inherent binding abilities of P1
and P2; (ii) the tertiary structure of the proteins can be
preserved upon transport across the interface as indicated by
the emission spectrum before and after equilibration; (iii) at
similar polymer and protein concentrations, the extent of
protein extraction in GFP (+15) is considerably higher than
GFP (−7), showing that binding affinity can influence the
extent of proteins transported across the interface.
In conclusion, we have demonstrated a set of supramolecular
assemblies, based on amphiphilic polymers, that can transport
proteins across a solvent interface. We have shown here that (i)
simple electrostatic complementarity in polymeric reverse
micelle systems can transport proteins from bulk aqueous
phase into the interior of a reverse micelle assembly in the
apolar organic phase; (ii) the activity of the transported
proteins is retained in the process; (iii) the efficiency of protein
binding is dependent on the charge density presented on the
protein surface; (iv) the kinetically trapped nature of the
assemblies suggest that the polymers do not ferry the proteins,
but instead transport likely occurs during the solvent exchange
within the interior of the assembly, when these assemblies
transiently find themselves at the interface during equilibration,
as illustrated in Figure 1; (v) specific ligand−receptor
interactions can be used to selectively extract proteins from
the aqueous phase. Overall, the most gratifying finding here is
that whole proteins can be moved across a solvent interface
into the interior of a supramolecular assembly, even though the
resident location of the assembly is in an incompatible solvent
for the protein. The preliminary findings here have implications
in many areas, especially in sensing, diagnostics, and catalysis.
For example, these systems can be further developed to detect
Though electrostatic complementarity can be utilized to
simplify protein mixtures and enable identification of the
presence of specific proteins, this ability will be greatly
enhanced if proteins can be transported across an interface in
response to a specific ligand−protein interaction. To investigate
this possibility, we used bovine carbonic anhydrase (bCA) as
the model protein, because aryl sulfonamides are well-
16
established as small molecule ligands for this protein. The
design hypothesis here is that if this ligand was installed in the
polymeric reverse micelles, it should be able to selectively
transport bCA to the organic phase due to specific binding.
For this purpose, we designed a zwitterionic amphiphilic
polymer P3 (Figure 5a), containing 40% decyl chain as the
hydrophobic moiety, 40% zwitterionic sulphobetain group as
the hydrophilic moiety and 20% benzenesulfonamide as the
ligand moiety. P3 forms a similar-sized assembly in apolar
solvents (Figure S3). When P3 was equilibrated with
tetramethylrhodamine-5-isothiocyanate (TRITC)-labeled
bCA, we observed a strong emission peak in organic phase,
indicating the transportation of TRITC−bCA conjugates.
Concurrently, there is a dramatic decrease in the fluorescence
intensity in the aqueous phase, indicating bCA is successfully
transported across the interface.
1
7−20
biomarkers in more complex mixtures of proteins.
Similarly, facile incorporation of active proteins in organic
solvents could facilitate enzyme-based catalysis for a broader
To investigate whether this is driven by the ligand-protein
binding, we designed a structurally similar amphiphilic polymer,
P4, which forms reverse micelles but lacks the sulfonamide
functional group. No change is observed in the emission
spectrum of both organic and aqueous phases, when using P4
as the transporter for TRITC−bCA (Figure 5b). To further test
whether the specific ligand-protein interaction is responsible for
the observed transport across the interface, we designed
another control experiment where the structure of the protein
was disrupted with acetonitrile and heat. The denatured bCA
should not be able to bind the sulfonamide ligands and thus
would not be transported into the organic phase. Indeed, no
fluorescence changes in the aqueous or organic phase are
observed, showing that no bCA was transported into the
organic phase (Figure 5b). These results confirm that
transportation occurs only when bCA’s native structure is
maintained in such a way to preserve its ability to bind the
sulfonamide ligand. Overall, these results suggest that specific
ligand-protein interactions can be utilized to bind and transport
proteins across the solvent interface.
21−25
range of organic substrates.
These constitute examples of
future directions for this research in our own laboratories.
ASSOCIATED CONTENT
■
*
S
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.7b13245.
Materials and methods, syntheses and characterization of
polymers, and supporting figures (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
*
*
rwvachet@chem.umass.edu
thai@chem.umass.edu
ORCID
Vincent M. Rotello: 0000-0002-5184-5439
Richard W. Vachet: 0000-0003-4514-0210
S. Thayumanavan: 0000-0002-6475-6726
Next, to test the ligand−protein binding based selectivity
associated with this process, we performed another set of
D
DOI: 10.1021/jacs.7b13245
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank NIH-NCI (CA-169140) for support.
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DOI: 10.1021/jacs.7b13245
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX