Mimicking multipass transmembrane proteins: Synthesis, assembly and folding of alternating amphiphilic multiblock molecules in liposomal membranes

Article abstract of DOI:10.1039/c0cc02420a

Alternating amphiphilic multiblock molecules 1-4, involving fluorescent hydrophobic units, were designed as mimics for multipass transmembrane proteins. Fluorescence spectroscopy of 1-4 in liposomal membranes suggested the face-to-face stacking of the hyd

Full text of DOI:10.1039/c0cc02420a

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Mimicking multipass transmembrane proteins: synthesis, assembly and
folding of alternating amphiphilic multiblock molecules in liposomal
membraneswz
a
a
b
b
Takahiro Muraoka, Tatsuya Shima, Tsutomu Hamada, Masamune Morita,
b
a
Masahiro Takagi and Kazushi Kinbara*
Received 7th July 2010, Accepted 27th August 2010
DOI: 10.1039/c0cc02420a
Alternating amphiphilic multiblock molecules 1–4, involving
fluorescent hydrophobic units, were designed as mimics for
multipass transmembrane proteins. Fluorescence spectroscopy
of 1–4 in liposomal membranes suggested the face-to-face
stacking of the hydrophobic units to give folded structures as
well as intermolecular assemblies.
introduced to both termini of 1–4. 1–4 tend to be soluble in
relatively polar organic solvents such as THF, DMF and
DMSO, while hardly soluble in water and non-polar solvents
such as hexane and CH Cl .
2
2
First, we investigated the aggregation behavior of BPEB
units of 1–4 in solution. The fluorescence spectra of 1–4 in
ꢁ5
THF (2.0 ꢀ 10 M) showed two emission peaks (lex = 313 nm)
in all cases, where no obvious difference was observed in the
spectral profiles among them (lem; 1: 367, 384 nm, 2: 366,
384 nm, 3: 367, 385 nm, 4: 367, 384 nm) (Fig. 2, blue lines).
These emission profiles indicate that the face-to-face stacking
Synthetic molecules mimicking the structure and function of
proteins have been attracting keen interest for development of
1
molecular devices and medicines. Membrane proteins are
2
important targets, where several synthetic ion channels,
3
4
9
including tubes, ribbons, and barrels as mimics of b-barrel
structures have been developed so far. On the other hand,
of BPEB units hardly occurs in THF. In sharp contrast, in a
ꢁ5
5
THF–water (1/9) mixture, 1–4 (2.0 ꢀ 10
M) showed
multipass transmembrane (MTM) proteins remain mostly
unexplored targets, although they provide one of the major
6
structural motifs of the membrane proteins. MTM proteins
red-shifted emission peaks in each case (lex = 313 nm,
em = 1: 426, 451 nm, 2: 427, 450 nm, 3: 450 nm, 4: 428,
l
452 nm) (Fig. 2, red lines). Moreover, absorption spectra of
1–4 showed a hypsochromic shift in a THF–water (1/9)
mixture compared with those in THF (see Supplementary
Information Fig. S1z). These spectral changes strongly indicate
that BPEB units form cofacial planar H-aggregates in aqueous
conditions. Dynamic light-scattering (DLS) analyses of 1–4
usually consist of a-helices connected by hydrophilic residues,
where their ternary structures are stabilized by the helix–helix
7
interaction. In this communication, as simple structural
mimics of MTM proteins, we report alternating amphiphilic
multiblock molecules 1–4, consisting of linearly connected
hydrophilic and hydrophobic moieties (Scheme 1). We found
that the hydrophobic units of 1–4, 1,4-bis(4-phenylethynyl)-
benzene (BPEB) units, tend to form face-to-face stacking in
ꢁ
5
(2.0 ꢀ 10 M) in a THF–water (1/9) mixture revealed the
presence of particles with average hydrodynamic diameters of ca.
100 nm in each case (see Supplementary Information Fig. S2z).
The aggregation behavior of 1–4 in the liposomal membranes
was then investigated using giant unilamellar liposomes of
8
the membrane, to possibly give folded structures like MTM
proteins (Fig. 1).
The BPEB unit is known to fluoresce at 390 nm upon
9
excitation around 320 nm. Since the face-to-face stacking of
BPEB units results in a bathochromic shift of the emission
band (lem = 440 nm), the assembling events of this unit are
able to be monitored by means of fluorescence spectroscopy.
BPEB units of 2–4 were connected by hydrophilic units
composed of a benzoate group bearing two tetraethylene
glycol chains. 1–4 were prepared by repeating Williamson
synthesis between phenolic OH and TEG tosylate (see
Supplementary Informationz). Finally, hydroxyl groups were
a
Institute of Multidisciplinary Research for Advanced Materials,
Tohoku University, 2-1-1, Katahira, Aoba-ku, Sendai 980-8577,
Japan. E-mail: kinbara@tagen.tohoku.ac.jp; Fax: +81 22-217-5612;
Tel: +81 22-217-5612
b
School of Materials Science, Japan Advanced Institute of Science
and Technology, 1-1, Asahidai, Nomi, Ishikawa 923-1292, Japan
w This article is part of the ‘Emerging Investigators’ themed issue for
ChemComm.
z Electronic supplementary information (ESI) available: Synthesis,
UV-Vis absorption spectra and profiles of dynamic light scattering
analyses of 1–4, and preparation of giant liposomes. See DOI:
10.1039/c0cc02420a
Scheme 1 Molecular structures of 1–4.
1
94 Chem. Commun., 2011, 47, 194–196
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Fig. 3 Optical micrographs of DOPC giant unilamellar liposomes
containing (a), (e) 1, (b), (f) 2, (c), (g) 3 and (d), (h) 4 ([1–4]/[DOPC] =
0
.10). (a)–(d) Phase-contrast and (e)–(h) fluorescence micrographs
(
lex; 330–385 nm, lobsd; >420 nm). Scale bars: 10 mm.
Fig. 1 Plausible models for the assemblies of 1 and 4 in a liposomal
membrane. (a) 1 and (c) 4 in dilute conditions. (b) 1 and (d) 4 in dense
conditions.
Fig. 4 Fluorescence spectra of 200 mM DOPC giant unilamellar
liposomes in water containing (a) 1, (b) 2, (c) 3 and (d) 4 with
excitation at 315 nm. [1–4]/[DOPC] = 0.00050, 0.0050, 0.050 and 0.10.
the membrane (Fig. 1a). However, of interest, when the
amount of 1 was increased, broad emission appeared at
>
400 nm with a decrease in the intensity of the original
emission. At molar ratio [1]/[DOPC] = 0.10, the spectrum
showed the maximum emission at 426 nm. Hence, the BPEB
unit of 1 is capable of assembling in the membrane to form
face-to-face stacking at relatively high concentrations
(Fig. 1b). In sharp contrast to 1, 4 in the membrane showed
emission bands at 428 and 448 nm (lex = 313 nm) even at very
low concentration ([4]/[DOPC] = 0.00050, Fig. 4d). Furthermore,
200-fold increase in the amount of 4 ([4]/[DOPC] = 0.10)
resulted in only a slight change in the spectral profile; the
fluorescence exhibited little dependency on the concentration
of 4. Thus, BPEB units of 4 are likely to form intramolecular
face-to-face stacking, allowing 4 to adopt a folded structure
like MTM proteins (Fig. 1c). However, slightly intensified
emission at lem > 480 nm (Fig. 4d) also suggests a
possible contribution of intermolecular stacking at higher
concentrations ([4]/[DOPC] > 0.0050) (Fig. 1d). Interestingly,
not only 4 but also 2 bearing two BPEB units showed emission
at 426 nm (lex = 313 nm) even at a highly diluted condition
Fig. 2 Fluorescence spectra of (a) 1, (b) 2, (c) 3 and (d) 4 in THF
(blue) and THF–water = 1/9 (red) with excitation at 313 nm.
1
,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), which can
0
1
be directly observed by optical microscopy.
Actually,
phase-contrast microscopy (200 mM DOPC containing 1–4,
1–4]/[DOPC] = 0.10) clearly visualized mm-sized unilamellar
liposomes in water (Fig. 3a–d). Of importance, these liposomes
[
were also visualized by fluorescence microscopy monitoring at
>420 nm (lex = 330–385 nm) (Fig. 3e–h), which demonstrates
successful incorporation of 1–4 into the liposomal membranes
under these conditions.
Fluorescence spectroscopy of DOPC liposomes prepared
with 1 in water (200 mM DOPC containing 1, [1]/[DOPC] =
0
.00050) showed emission peaks at 366 and 384 nm
(
lex = 315 nm, Fig. 4a). The spectral profile is quite similar
to that of 1 in THF (Fig. 2a, blue line). Namely, at this
concentration, 1 does not form the face-to-face assemblies in
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Chem. Commun., 2011, 47, 194–196 195

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(
(
[2]/[DOPC] = 0.00050), while 3 showed red-shifted emission
425 nm) only at high concentrations ([3]/[DOPC]
.050–0.10). This apparent even–odd effect may suggest
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formation of dimeric assemblies of BPEB units, which play
some roles in folding and assembling of 1–4 in the membrane.
It is worthy of note that in spite of intermolecular assembling
of 1–4 in the membrane, no mm-scale domain structure like
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In conclusion, the BPEB unit was found to have strong
capabilities to be involved in a liposomal membrane and form
face-to-face stacking. These features could be applied for
controlled folding of molecules as was found for alternating
amphiphilic multiblock molecules 2 and 4, which can be
regarded as structural mimics of two- and four-transmembrane
proteins, respectively. MTM proteins exhibit diverse functions,
such as cell-adhesion of four-transmembrane proteins,
2
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We thank Prof. M. Shimomura and Dr T. Higuchi for
assistance in DLS measurements. We also thank Ms Yuko
Kishimoto for support in preparation and microscopy of
liposomes. This work was performed under the Cooperative
Research Program of ‘‘Network Joint Research Center for
Materials and Devices (Institute of Multidisciplinary Research
for Advanced Materials, Tohoku University)’’, and partially
supported by the Ministry of Education, Science, Sports and
Culture, Japan, Grant-in-Aid for Young Scientists S and
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96 Chem. Commun., 2011, 47, 194–196
This journal is c The Royal Society of Chemistry 2011