Archaea analogue thiolipids for tethered bilayer lipid membranes on ultrasmooth gold surfaces

Article abstract of DOI:10.1002/anie.200390080

Simple vesicle fusion allowed the formation of a bilayer lipid membrane from the self-assembled monolayer of lipoic acid ester 1 (see formula). Film thicknesses determined by surface plasmon resonance (SPR) spectroscopic measurements agree with the theore

Full text of DOI:10.1002/anie.200390080

Communications
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ratory, Richland, WA, USA, and funded by the U.S. Department
of Energy.
+N(CH3
)
3
+N(CH3
)
+N(CH3
)
+N(CH3
)
3
+N(CH3
)
3
+N(CH3
)
3
3
+N(CH3
O
)
3
3
O
O
O
O
O
O
O
O
O
O
O-
O
P
O
O-
P
O
O-
P
O-
O
P
P
O-
O
O-
P
P
O-
O
O
O
O
[13] To rationalize the different steric requirements for the horizon-
tally and vertically arranged borylene ligand a ™f-y∫ search was
performed. In this the dihedral angles H-C-Si-N, C-Si-N-B, and
C-Cpm-V-B were changed in 58 steps over a range of 1208 for the
former two and 728 for the latter one, and the distances between
terminal atoms of different groups, that is Cp, Me, and CO were
calculated. For geometries with the smallest interatomic dis-
tances the search was repeated in 28 steps over ranges of ꢁ 608
and ꢁ 368. The following distances [ä] were determined;
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
3.5 nm
O
O
ꢀ
vertical: Cp Me 1.633 (smallest distance), 2.412 (average
ꢀ
distance), CO Me 1.652 (smallest distance), 2.308 (average
O
ꢀ
O
O
O
O
distance); horizontal: Cp Me 1.959 (smallest distance), 2.887
(average distance), CO Me 1.214 (smallest distance), 1.926
(average distance). While Cp Me distances are comparable and
even somewhat larger for the horizontal orientation, the very
O
O
O
O
O
O
O
O
O
ꢀ
4.7 nm
O
O
O
O
O
O
O
ꢀ
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
ꢀ
small CO Me distances for the latter are evident. Although
O
O
O
O
O
those results are of qualitative nature, they clearly provide
evidence that the horizontal arrangement is sterically less
favored.
O
O
O
O
O
O
O
O
O
O
O
O
S
S
S
S
S
S
S
S
S
S
S
S
S
S
0.5 nm
[14] C. K. Fair, MolEN, An Interactive Intelligent System for Crystal
Structure Analysis, Enraf-Nonius, Delft, The Netherlands, 1990.
[15] G. M. Sheldrick, Program Package SHELXTL, Version 5.1.
Bruker Analytical X-ray Systems, Inc., Madison, Wisconsin,
USA, 1998.
Au
LaSFN9
Figure 1. Schematic representation of the arrangement of the DPTL
molecules to form the tBLMs. Molecular dimensions are in proportion
to the ultrasmooth gold surface to guarantee the perfect alignment of
the phytanyl chainsin the formation of a dense mono- and bilayer. The
tetraethylene glycol chains are assumed to be stretched.
LaSFN9¼high-refractive-index glass.
Model Systems for Membranes
a lipid group linked to a metal surface, preferably gold,
through a thiol-modified tether as spacer unit between the
lipid and the gold substrate. The tether/spacer molecules
should decouple the membrane from the surface and thus
provide a hydrophilic layer (between the membrane and the
substrate) with the functional properties of the cytosol/
cytoskeleton.[1] They should establish a water-containing
submembrane space, which reduces the hydrophobic influ-
ence of the metal surface,[2] thus enabling the functional
incorporation of membrane proteins.[3]
Archaea Analogue Thiolipids for Tethered
Bilayer Lipid Membranes on Ultrasmooth Gold
Surfaces**
Stefan M. Schiller,* Renate Naumann,
Katherine Lovejoy, Horst Kunz, and
Wolfgang Knoll
Moreover, the combination of such ultrathin tBLMs with
the robustness of a gold support in a planar configuration
allows the application of surface-sensitive techniques, includ-
ing surface plasmon resonance spectroscopy (SPR), measure-
ments with a quartz-crystal microbalance (QCM), surface-
enhanced IR, and electrochemical techniques such as electro-
chemical impedance spectroscopy (EIS). The metal surface
also allows the investigation of surface molecules under a
defined electric field, thus extending the scope of experimen-
tal conditions significant to the study of field-sensitive
processes, for example, ion transport through receptors,
channels, and carriers such as valinomycin.
Tethered bilayer lipid membranes (tBLMs) mimick the
biological cytoplasmic membrane and are promising tools
for basic research and biosensor applications. They comprise
[*] Dipl.-Chem. S. M. Schiller, Dr. R. Naumann, Prof. Dr. W. Knoll
Max-Planck-Institut f¸r Polymerforschung
Ackermannweg 10, 55128 Mainz (Germany)
Fax: (þ49)6131-379-100
E-mail: schiller@mpip-mainz.mpg.de
K. Lovejoy
Department of Chemistry, Northwestern University
Evanston, IL 60208-3113 (USA)
The tBLMs reported so far are supplemented with polar
Dipl.-Chem. S. M. Schiller, Prof. Dr. H. Kunz
Institut f¸r Organische Chemie, Universit‰t Mainz
Duesbergweg 10 24, 55128 Mainz (Germany)
8]
peptide,[4] oligomer,[5] or polymer[6 spacers. Poly- or oligo-
meric tether molecules based on poly- or oligo(ethylene
glycol) modified with dialkyl lipids tails have been employed
in many cases. However, the capacitance and impedance of
these tBLMs were found in the ranges 0.5 0.7 mFcmꢀ2 and
0.01 0.1 MWcm2, respectively,[5,6,9,10] insufficient to match the
electrical properties of biological membranes. The model
[**] We thank Iris K‰rcher for skillful technical assistance and Krasimir
Vasilev for the preparation of template-stripped gold (TSG). K.L. was
a summer student at the MPIP and was supported by the Center for
Polymer Interfaces and Macromolecular Assemblies (CPIMA) under
NSF grant (DMR-9808677).
208
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Angewandte
Chemie
another key feature, the application of the ultrasmooth
O
O
O
O
O
surface of template-stripped gold (TSG),[19] to form tBLMs
with good insulating properties. Smooth metal surfaces such
as that of mercury have been shown to be effective for the
formation of tBLMs.[20]
O
O
O
S
S
surface binding
residue
polar tether group
hydrophobic ether linked
isoprenoid tails
The roughness of TSG was shown to be 0.5 nm over areas
of mm2 by AFM, one order of magnitude smaller than the
molecular dimension of the DPTL molecule itself (Figure 1).
This ensures the perfect supramolecular alignment of the
functional units, resulting in a perfect arrangement of the
monolayer. For comparison, the roughness of polycrystalline
gold often used for the preparation of SAMs can be as high as
5 10 nm. Au (111), obtained by annealing the gold surfaces,
has a roughness of 5 nm over an area of several mm2
(measured by AFM, not shown). Rough surfaces seem to be
a cause of the low impedance values observed in many model
membranes.
system that best reflects the electrical properties of the
biomembrane is the bilayer lipid membrane (BLM), which
shows capacitance values of around 0.5 mFcmꢀ2 and impe-
dance values of ꢂ 10 MWcm2.[11,12] tBLMs with good elec-
trical insulation properties were presented by Cornell
et al.,[13] who used a multicomponent system consisting of a
monophytanoyl spacer lipid together with a transmembrane
lipid.
In contrast, the tBLM presented herein is based on a
single molecule, the novel archaea analogue 2,3-di-O-phy-
tanyl-sn-glycerol-1-tetraethylene glycol-d,l-a-lipoic acid es-
ter lipid (DPTL). Phytanyl chains were chosen as hydro-
phobic tails instead of alkyl chains because of their low phase-
transition temperature and their influence on the density and
stability of biological membranes.[14] The
High resistive tBLMs have been prepared from a SAM of
DPTL on the TSG surface. The fusion of liposomes prepared
from diphytanoylphosphatidylcholine (DPhyPC) gave rise to
the formation of well-defined lipid bilayers as shown by SPR
Table 1: Data obtained by simulations from EIS (capacitance and resistance, Figure 2) and SPR
(thickness) measurements. Molecule sizes obtained by modeling calculations with CS Chem3D Pro are
added for comparison.
phase-transition temperature of 2,3-di-O-
phytanyl-sn-glycerol-1-tetraethylene glycol
was determined to be < ꢀ808C by differ-
ential scanning calorimetry (DSC). In con-
trast to the system of Cornell et al.[13] which
bears one phytanoyl chain, our system has
two phytanyl chains bound to the spacer
through a chiral glycerol unit since it is
known that a single hydrophobic chain does
not guarantee a stable insertion of molecules
into a lipid membrane.[15] Furthermore, the
2,3-di-O-phytanyl-sn-glycerol unit contains
only ether linkages to prevent hydrolytic
cleaving. This moiety is known to form stable
biomembranes under the extreme living
conditions (e.g. high temperatures) of ex-
tremophiles or archaea.[16] The importance
of the ether linkage and the absence of esters
and polar phosphatidyl groups to form
highly impermeable membranes was exper-
imentally demonstrated by Mathai et al.[17]
According to FTIR spectroscopic stud-
ies, the tetraethꢀylene glycol spacer can be
considered as an elongated hydrophilic chain
(Figure 1), which provides the maximum
decoupling distance of the lipid membrane
from the substrate. Other oligoethylene
glycol (OEG) moieties such as penta- to
octa(ethylene glycol) moieties adopt a 7/2
helical structure; the octa(ethylene glycol)
appears to have a slightly distorted helical
conformation.[18] The synthesis of DPTL is
illustrated in Scheme 1.
Cll (exp.)
Rll (exp.)
[mWcm2]
d (exp.)
[nm]
d (calcd)
[nm]
[mFcmꢀ2
]
DPTL, monolayer before vesicle spreading
lipid bilayer after vesicle spreading
lipid bilayer in KCl (0.1m) þ valinomycin
lipid bilayer in KCl (0.1m) þ valinomycin after
washing with NaCl (0.1m)
0.55
0.49
0.64
0.61
1.75
4.35
0.0022
0.31
4.7
8.5
4.7
8.2
HO
O
O
O
c)
O
O
a)
TosO
O
O
b)
O
4
OH
4
4
O
O
OH
O
4
1
3
2
d)
Br
5
O
O
HO
O
4
O
O
4
O
e)
O
6
7
f)
O
O
O
O
O
O
O
O
S S
DPTL
Scheme 1. Synthesis of DPTL: Acetal 1 was synthesized from d-mannitol according to reference [19].
a) Tetra(ethylene glycol) (20 equiv), NaH (2.5 equiv), dioxane, DMAP (cat.), Ar, 45 758C, 3 d; b) NaH
(2 equiv), BnBr (2.5 equiv), TBAI (cat.), DMF, Ar, 0!208C, 2 d, 35% (over steps a) and b)); c) H2O/
THF, Dowex50WX8, 508C, 18 h, 89%; d) NaH (2.1 equiv), 5 (4 equiv, synthesized from phytol
according to reference [20]), DMF, Ar, room temperature,4 d, 49%; e) THF, MeOH, H2, Pd/C
(0.3 equiv), room temperature, 30 min, quant.; f) lipoic acid (5 equiv), EDC (6.6 equiv), DMAP (cat.),
CH2Cl2, Ar, 1 d, 60%. Bn¼benzyl, TBAI¼tetrabutylammonium iodide, DMF¼N,N-dimethylform-
amide, EDC¼1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, DMAP¼4-dimethylami-
The influence of structure and morphol-
ogy of the gold substrate on the preparation
of self-assembled monolayers (SAMs) is
often disregarded. Herein we introduce nopyridine.
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Communications
and EIS measurements. We have found only recently that
SPR and EIS can be applied simultaneously not only to
polycrystalline gold[21] but also to the TSG surface, provided
that an optical glue is used for the preparation.[21] Hence this
powerful combination of techniques was used to investigate
the formation of tBLMs based on the DPTL molecule. Layer
thicknesses were calculated from SPR measurements on the
basis of a three-layer system of glass, gold, and the lipid layer.
For the latter, a refractive index of n ¼ 1.5 is assumed and the
glue was assumed to have the same refractive index as glass.
Results are given in Table 1. The thickness values are in
agreement with the dimensions of the molecules, which were
modeled with CS Chem3D Pro (CambridgeSoft Corporation,
Version 4.0). The configuration represented in Figure 1 shows
a slight tilt angle of the molecular axis and the extended
conformation of the OEG spacer. The increase in the
thickness of the layer by fusion of the liposomes was
monitored in real time by means of SPR spectroscopy and
clearly showed saturation within 60 min, which indicates the
unrolling of vesicles on a hydrophobic surface to form a lipid
bilayer.
The impedance spectra are shown in Figure 2. From these
spectra, the capacitance and the resistance of the lipid mono-
and bilayer were calculated by fitting the data to the
equivalent circuit.[22,24,25] The circuit consists of a RC mesh
representing the lipid film, the resistance of the electrolyte
solution, and the capacitance of the diffuse double layer
adjacent to the gold. The results are given in Table 1. Data
measured for the tBLMs varied from 0.45 to 0.8 mFcmꢀ2 for
the capacitance and from 2 to 12 MWcm2 for the resistance.
They thus compare quite well with those of the BLMs.
Also shown in Figure 2 is the effect of valinomycin on the
resistance of the tBLM in the presence of potassium ions.
Valinomycin is a potassium ionophore antibiotic, produced by
Streptomyces fulvissimus,[26] which specifically includes potas-
sium ions in its hydrophilic interior, while the hydrophobic
exterior enables transmembrane transport of the potassium
ions. This gives rise to the so-called inflection point in the EIS
spectrum, at which point the resistance drops to the kW range
(Table 1) and the capacitance of the diffuse double layer
evolves to approximately 5 mFcmꢀ2, in agreement with values
reported by other authors.[22,24,25]
It is concluded from these experiments that tBLMs can be
prepared on SAMs of DPTL simply by vesicle fusion; the
molecules are arranged almost perpendicular to the surface,
particularly on ultrasmooth gold surfaces. The obtained
tBLMs are stable over days and have electrical properties
that resemble those of the biological membrane. The tetra-
ethylene glycol spacer allows the formation of a diffuse
double layer in the aqueous space between the lipid layer and
the substrate, thus mimicking the cytoskeleton.[22,24] The
tBLM presented herein can thus be seen as model system
for biological membranes.
Figure 2. EIS analyses of TSG films coated with DPTL monolayers be-
*
fore (&) and after ( ) fusion with DPhyPC liposomes in KCl solution
(0.1 molLꢀ1), the latter after adding valinomycin (0.1 mm) (hexagons),
and after washing with NaCl (0.1 molLꢀ1) ( ). The solid lines repre-
~
sent fitted data. A) Z’ (real part of the impedance) versus n (frequen-
cy); B) q (phase shift or phase angle) versus n (frequency); C) equiva-
lent circuit used for fitting purposes.
the mica sheets at 6508C for 45 s to form Au(111). The gold surface
was then glued with EPO-TEK 377 (Polytec GmbH, Waldbronn,
Germany) to high-refractive-index (n ¼ 1.7) glass slides and heated at
1508C for 60 min. After cooling, these slides were immersed in THF
for 30 s to allow the mica sheets to be separated from the gold film,
which were then thoroughly rinsed with ethanol.
Experimental Section
Au(111) surfaces and TSG: 60-nm gold films were deposited by
electrothermal evaporation (0.01 0.05 nmsꢀ1
,
2 î 10ꢀ6 mbar) on
Formation of SAMs and vesicle fusion: TSG slides were placed
for 24 h in an ethanolic solution of DPTL (0.2 mgmLꢀ1), rinsed in
freshly cleaved mica sheets. The gold surface was annealed by heating
210
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Angewandte
Chemie
pure ethanol, and dried under a stream of nitrogen. The prepared
slides were then mounted in a measuring cell described earlier[23] for
simultaneous SPR and EIS experiments. The thickness of the SAMs
were measured in a solution of KCl (0.1 molLꢀ1) before and after the
addition of DPhyPC vesicles (DPhyPC supplied from Avanti Polar
Lipids, Inc., Alabaster, Al, USA) freshly prepared by extrusion
through a 50-nm polycarbonate filter. Fusion of vesicles was carried
out at 308C at a final concentration of 0.02 mgmLꢀ1. EIS spectra were
taken before and after the addition of valinomycin (final concen-
tration ¼ 4 î 10ꢀ6 molLꢀ1). Surface plasmons were excited by a 632.5-
nm HeNe laser. EIS measurements were conducted at a bias potential
of 0 V vs. Ag/AgCl, saturated NaCl reference electrode and a
platinum counter electrode.
[12] a) R. Guidelli, G. Aloisi, L. Becucci, A. Dolfi, M. R. Moncelli,
F. T. Buoninsegni, J. Electroanal. Chem. 2001, 504, 1; b) W.
Knoll, C. W. Frank, C. Heibel, R. Naumann, A. Offenh‰user, J.
R¸he, E. K. Schmidt, W. W. Shen, A. Sinner, Rev. Mol.
Biotechnol. 2000, 74, 137; c) E. K. Sinner, W. Knoll, Curr. Opin.
Chem. Biol. 2001, 5, 705.
[13] B. A. Cornell, V. L. B. Braach-Maksvytis, L. G. King, P. D. J.
Osman, B. Raguse, L. Wieczorek, R. J. Pace, Nature 1997, 387,
580.
[14] V. L B. Braach-Maksvytis, B. Raguse, J. Am. Chem. Soc. 2000,
122, 9544.
[15] R. M. Epand, Biopolymers 1997, 43, 15.
[16] C. R. Woese, G. E. Fox, Proc. Natl. Acad. Sci. USA 1977, 74,
5088.
[17] J. C. Mathai, G. D. Sprott, M. L. Zeidel, J. Biol. Chem. 2001, 276,
27266.
Received: August 8, 2002 [Z19921]
[18] D. J. Vanderah, C. P. Pham, S. K. Springer, V. Silin, C. W. Meuse,
Langmuir 2000, 16, 6527.
[19] W. J. Kerr, G. G. Kirk, D. Middlemiss, J. Organomet. Chem. 1996,
519, 93.
[1] E. Sackmann, Science 1996, 271, 43.
[2] W. J. Miller, N. L. Abbott, Langmuir 1997, 13, 7106.
[3] R. Naumann, E. K. Schmidt, A. Jonczyk, K. Fendler, B.
Kadenbach, T. Liebermann, A. Offenh‰usser, W. Knoll, Biosens.
Bioelectron. 1999, 14, 651.
[4] E. K. Schmidt, T. Liebermann, M. Kreiter, A. Jonczyk, R.
Naumann, A. Offenh‰usser, E. Neumann, A. Kukol, A. Mae-
licke, W. Knoll, Biosens. Bioelectron. 1998, 13, 585.
[5] H. Lang, C. Duschl, H. Vogel, Langmuir 1994, 10, 197.
[6] M. L. Wagner, L. K. Tamm, Biophys. J. 2000, 79, 1400.
[7] C. Erdelen, L. H‰ussling, R. Naumann, H. Ringsdorf, H. Wolf, J.
Yang, M. Liley, J. Spinke, W. Knoll, Langmuir 1994, 10, 1246.
[8] J. Spinke, J. Yang, H. Wolf, M. Liley, H. Ringsdorf, W. Knoll,
Biophys. J. 1992, 63, 1667.
[20] a) M. Ishiwatari, K. Yamada, R. Ishiwatari, Chem. Lettl 2002, 3,
206; b) P. G. Schouten, J. F. Van der Pol, J. W. Zwikker, W.
Drenth, S. J. Picken, Mol. Cryst. Liq. Cryst. 1991, 195, 291.
[21] P. Wagner, M. Hegner, H. J. G¸ntherodt, G. Semenza, Langmuir
1995, 11, 3867.
[22] C. Peggion, F. Formaggio, C. Toniolo, L. Becucci, M. R.
Moncelli, R. Guidelli, Langmuir 2001, 17, 6585.
[23] N. Bunjes, E. K. Schmidt, A. Jonczyk, F. Rippmann, D. Beyer, H.
Ringsdorf, P. Gr‰ber, W. Knoll, R. Naumann, Langmuir 1997, 13,
6188.
[24] B. Raguse, V. L B. Braach-Maksvytis, B. A. Cornell, L. G. King,
P. D. D. Osman, R. J. Pace, L. Wieczorek, Langmuir 1998, 14,
648.
[9] S. Heyse, O. P. Ernst, Z. Dienes, K. P. Hofmann, H. Vogel,
Biochemistry 1998, 37, 507.
[25] G. Krishna, J. Schulte, B. A. Cornell, R. Pace, L. Wieczorek, P. D.
Osman, Langmuir 2001, 17, 4858.
[26] H. Neurath, R. L. Hill, The Proteins, Vol. 5, Academic Press,
New York, 1982.
[10] L. M. Williams, S. D. Evans, T. M. Flynn, A. Marsh, P. F.
Knowles, R. J. Bushby, N. Boden, Langmuir 1997, 13, 751.
[11] G. Wiegand, N. Arribas-Layton, H. Hillebrand, E. Sackmann, P.
Wagner, J. Phys. Chem. B 2002, 106, 4245.
Angew. Chem. Int. Ed. 2003, 42, No. 2
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