Phospholipid bicelles that align with their normals parallel to the magnetic field

Article abstract of DOI:10.1021/ja027079n

We have recently reported phospholipid bicelles (bilayered micelles) that have positive anisotropy of the magnetic susceptibility and align with their normals parallel to an external magnetic field [J. Am. Chem. Soc. 2001, 123, 1537]. Improvements have been made via the synthesis of a new phospholipid, 1-dodecanoyl-2-(4-(4-biphenyl)butanoyl)-sn-glycero-3-phosphocholine (DBBPC). Bicelles can be formed by mixing DBBPC with a short-chain phospholipid, 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) in a ratio between 5.1:1 and 6.5:1 in an aqueous medium. The ³¹P NMR spectra clearly show that these bicelles align with their principal axes parallel to the magnetic field within a wide temperature range. The ³¹P chemical shifts indicate that the conformation of the polar headgroup in these bicelles may be different from that in common bicelles. The phase behavior of a mixture of DBBPC/DHPC with 6:1 mole ratio was investigated in the temperature range of 10-75 °C using ³¹P, ²H, and ²³Na NMR. At lower temperatures (10-54 °C), the system is dominated by the bicellar phase. At higher temperatures (54-75 °C), isotropic micelles are formed and coexist with the bicelles. The partial alignment of maltotriose in the DBBPC/ DHPC system was studied at three temperatures, and the ¹H-¹³C dipolar coupling constants are compared with those obtained for two other bicelle solutions.

Full text of DOI:10.1021/ja027079n

Published on Web 09/10/2002
Phospholipid Bicelles That Align with Their Normals Parallel
to the Magnetic Field
Chibing Tan,† B. M. Fung,*,† and Gyoujin Cho‡
Contribution from the Department of Chemistry and Biochemistry, UniVersity of Oklahoma,
Norman, Oklahoma 73019-3051, and Department of Chemical Engineering,
Sunchon National UniVersity, 315 Maegok Sunchon, Chonnam 540-742, Korea
Received May 28, 2002
Abstract: We have recently reported phospholipid bicelles (bilayered micelles) that have positive anisotropy
of the magnetic susceptibility and align with their normals parallel to an external magnetic field [J. Am.
Chem. Soc. 2001, 123, 1537]. Improvements have been made via the synthesis of a new phospholipid,
1-dodecanoyl-2-(4-(4-biphenyl)butanoyl)-sn-glycero-3-phosphocholine (DBBPC). Bicelles can be formed
by mixing DBBPC with a short-chain phospholipid, 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC)
in a ratio between 5.1:1 and 6.5:1 in an aqueous medium. The 31P NMR spectra clearly show that these
bicelles align with their principal axes parallel to the magnetic field within a wide temperature range. The
31P chemical shifts indicate that the conformation of the polar headgroup in these bicelles may be different
from that in common bicelles. The phase behavior of a mixture of DBBPC/DHPC with 6:1 mole ratio was
2
investigated in the temperature range of 10-75 °C using 31P, H, and 23Na NMR. At lower temperatures
(10-54 °C), the system is dominated by the bicellar phase. At higher temperatures (54-75 °C), isotropic
micelles are formed and coexist with the bicelles. The partial alignment of maltotriose in the DBBPC/
DHPC system was studied at three temperatures, and the 1H-13C dipolar coupling constants are compared
with those obtained for two other bicelle solutions.
Introduction
can be better described as a heavily perforated, highly dynamic
lamellar bilayer phase. In other words, the bilayers resemble
Phospholipid (PC) bicelles were initially described as disklike
bilayered micelles.1 They consist of an appropriate combination
of long-chain phospholipid, such as 1,2-ditetradecanoyl-sn-
glycero-3-phosphocholine (DMPC, where M stands for myris-
toyl) or 1,2-didodecanoyl-sn-glycero-3-phosphocholine (DLPC,
where L stands for lauroyl, 2), and a short-chain phospholipid,
such as 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC, 1),
in an aqueous medium. Bicelles exhibit typical lyotropic liquid
crystalline properties with in a limited temperature range.1a,2 It
was suggested that the long-chain PC forms the plane of the
disk and the short-chain PC forms the edge.1a,3 The long-chain
PC can be replaced by other phospholipids to increase the
chemical stability4 or impart a charge5 on the bicelles. Recently,
based on the results of small-angle neutron scattering6 and
translational diffusion anisotropy measurements of a probe
molecule,7 it was suggested that the morphology of the bicelles
slices of Swiss cheese, a picture first suggested for the bicelles
when (and only when) Tm3+ is added to the system.8 In this
description, the rims surrounding the holes in the bilayer consist
of DHPC,7 and the holes are probably dynamic rather than static.
The perforated lamellar phase (previously considered to be
nematic) of bicelles can be macroscopically aligned in a
magnetic field, and this has been used extensively for nuclear
magnetic resonance (NMR) studies of biological molecules. The
magnetically aligned bicelles are also useful in low-angle X-ray
and neutron diffraction studies.6 Initially, the emphasis was on
the use of bicelles as model membranes for NMR studies of
membrane-associated peptides and proteins.1,9 Since the publica-
tion of the seminal work by Bax and Tjandra,10 the use of dilute
bicelle solutions as ordering media for water-soluble proteins
and DNA has found wide application.
Common PC bicelles have a negative anisotropy of the
magnetic susceptibility (∆ø), so that their normals align in all
directions perpendicular to the external field, spanning a 360°
distribution. Consequently, the NMR peaks of species dissolved
in the interior or bound to the exterior might show partial powder
* Address correspondence to this author. E-mail: bmfung@ou.edu.
† University of Oklahoma.
‡ Sunchon National University.
(1) (a) Sanders, C. R.; Schwonek, J. P. Biochemistry 1992, 31, 8898. (b)
Sanders, C. R. Chem. Phys. Lipids 1994, 72, 41. (c) Sanders, C. R.; Hare,
B. J.; Howard, K. P.; Prestegard, J. H. Prog. NMR Spectrosc. 1994, 26,
421. (d) Sanders, C. R.; Prosser, R. S. Structure 1998, 6, 1227.
(2) Sternin, E.; Nizza, D.; Gawrisch, K. Langmuir 2001, 17, 2610.
(3) (a) Vold, R. R.; Prosser, R. S. J. Magn. Reson. B 1996, 113, 267. (b)
Struppe, J.; Vo1d, R. R. J. Magn. Reson. 1998, 135, 541.
(4) (a) Ottiger, M.; Bax, A. J. Biomol. NMR 1998, 12, 361. (b) Cavagnero, S.;
Dyson, H. J.; Wright, P. E. J. Biomol. NMR 1999, 13, 387.
(5) Losonczi, J. A.; Prestegard, J. H. J. Biomol NMR 1998, 12 (3), 447.
(6) Nieh, M. P.; Glinka, C. J.; Krueger, S.; Prosser, R. S.; Katsaras, J. Langmuir
2001, 17, 2629.
(7) Gaemers, S.; Bax, A. J. Am. Chem. Soc. 2001, 123, 12343.
(8) Prosser, R. S.; Hwang, J. S.; Vold, R. R. Biophys. J. 1998, 74, 2405.
(9) (a) Sanders, C. R.; Landis, G. C. J. Am. Chem. Soc. 1994, 116, 6470. (b)
Sanders, C. R.; Landis, G. C. Biochemistry 1995, 34, 4030. (c) Howard,
K. P.; Opella, S. J. J. Magn. Reson. B 1996, 112, 91. (d) Vold, R. R.;
Prosser, R. S.; Deese, A. J. J. Biomol. NMR 1997, 9, 329.
(10) (a) Tjandra, N.; Bax, A. Science 1997, 278, 1111. (b) Bax, A.; Tjandra, N.
J. Biomol. NMR 1997, 10, 289.
9
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J. AM. CHEM. SOC. 2002, 124, 11827-11832
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A R T I C L E S
Tan et al.
Scheme 1. Synthetic Scheme for DBBPC
patterns, limiting the usefulness of the NMR studies. Further-
more, because the magnetic susceptibility anisotropy of phos-
pholipids and of the R-helical protein segments have opposite
signs, the addition of proteins containing membrane-spanning
R-helices may cancel the tendency for the bilayers to align well.8
Although the direction of alignment can be changed with
variable-angle rapid sample spinning,11 a uniform alignment of
the bicelle axes along the direction of the magnetic field is most
desirable. For these reasons, efforts have been made to construct
bicelles that would align with their normals parallel to the
external field and have a uniform alignment. For the study of
water-soluble species, the direction of alignment of the bicelles
is not critical because they function only as an ordering matrix.
However, in many cases it is very beneficial to carry out the
NMR study in media with two different alignments12 so that
the data can be analyzed to yield more detailed structural
information. Therefore, PC bicelles that align with their normals
parallel to the magnetic field will also be useful in these cases.
Two approaches had been used to change the alignment of
the bicelle normal from perpendicular to parallel. The first
method is to use amphiphilic aromatic compounds to mix with
phosphatidylcholine13 because the phenyl ring has a large
positive ∆ø,14 but the amount of the additive required is large
and the results are usually not very satisfactory. The second
method is to use paramagnetic lanthanide ions to change the
sign of ∆ø to make the bicelles turn around,8,15 but the effect
of the paramagnetic ions is a concern. To overcome these
problems, we developed a modified PC (DBPC, 3), in which
one of the long aliphatic chains was replaced by a biphenyl
moiety, to formulate PC bicelles without using other additives.16
It was shown that mixtures of DBPC/DHPC with a ratio very
close to 6 can form bicellar solutions which are stable from 8
to 40 °C. Because the two phenyl rings at DBPC impart a large
positive ∆ø on the bicelles, they do “flip over” in a magnetic
field to achieve the desired magnetic alignment. Subsequently,
we have made an improvement on this system by synthesizing
another PC that also contains a biphenyl unit but a longer spacer,
1-dodecanoyl-2-(4-(4-biphenyl)-butanoyl)-sn-glycero-3-phos-
phocholine (DBBPC, 4). The phase behavior of mixtures formed
by mixing this new PC and DHPC was investigated by using
31P, 23Na, and 2H NMR spectroscopy. It was found that bicelles
are formed in a wider range of composition and with better
thermal stability. The results are reported here.
Experimental Section
DLPC, DHPC, and 1-lauroyl-2-hydroxy-sn-glycero-3-phosphocho-
line were purchased from Avanti Polar Lipids Co.; all other compounds
were purchased from Aldrich Chemicals. DLPC rather than DMPC
was used because the bicellar phase begins to form at a lower
temperature. These starting materials were used directly without further
purification. The synthetic scheme is shown in Scheme 1, and the
procedures are described in the following.
(11) Zandomeneghi, G.; Tomaselli, M.; Beek, J. D.; Meier, B. H. J. Am. Chem.
Soc. 2001, 123, 910.
(12) Al-Hashimi, H. M.; Majumdar, A.; Gorin, A.; Kettani, A.; Skripkin, E.;
Patel, D. J. J. Am. Chem. Soc. 2001, 124 (4), 633.
4-(4-Biphenyl)-4-oxobutanoic Acid.17 To a 100 mL round-bottom
flask containing 3.0 g (30 mmol) of succinic anhydride and 5.0 g (32
mmol) of biphenyl 30 mL of nitrobenzene was added. The mixture
was heated until a clear solution was obtained and then cooled to room
temperature. The solution became opaque at room temperature. Then,
8.0 g (60 mmol) of powdered anhydrous aluminum chloride was added
slowly while the mixture was being stirred, taking about 5 min. The
solution became clear and finally turned black. After the stirring was
(13) Sanders, C. R.; Schaff, J. E.; Prestegard, J. H. Biophys. J. 1993, 64, 1069.
(14) Sakurai, I.; Kawamura, Y.; Ikegami, A.; Iwayanagi, S. Proc. Natl. Acad.
Sci. U.S.A. 1980, 77, 7232.
(15) (a) Prosser, R. S.; Hunt, J. A.; Dinatale, J. A.; Vold, R. R. J. Am. Chem.
Soc. 1996, 118, 269. (b) Katsaras, J.; Donaberger, R. L.; Swainson, J. P.;
Tennant, D. C.; Tun, Z.; Vold, R. R.; Prosser, R. S. Phys. ReV. Lett. 1997,
78, 899.
(16) Cho, G.; Fung, B. M.; Reddy, V. B. J. Am. Chem. Soc. 2001, 123, 1537.
(17) Furniss, B. S., Hannaford, A. J., Smith, P. W. G., Tatchell, A. R., Revisers;
Vogel’s Textbook of Practical Organic Chemistry, 5th ed.; Longman
Scientific & Technical: Essex, UK, 1989.
9
11828 J. AM. CHEM. SOC. VOL. 124, NO. 39, 2002

Properties of Phospholipid Bicelles
A R T I C L E S
continued for 24 h at room temperature with an HCl trap, the reaction
was stopped by adding 15 mL of water and 5 mL of concentrated
hydrochloric acid. The yellow precipitate was filtered and dried, and
then washed thoroughly with ether. The crude product was purified
using silica column chromatography (chloroform/acetone 2:1 as eluent)
to obtain 4-(4-biphenyl)-4-oxobutanoic acid as a white solid, with 70%
yield. 1H NMR (400 MHz, CDCl3): δ 7.6 (d, 2H, Ar), 7.5 (d, 2H, Ar),
7.4 (m, 2H, Ar), 7.3 (m, 1H, Ar), 7.24 (d, 2H, Ar), 5.15 (m, 1H, CH),
2.7 (m, 2H, CH2), 2.4 (m, 2H, CH2), 2.0 (m, 2H, CH2).
4-(4-Biphenyl)butanoic Acid.17 Amalgamated zinc was prepared
from zinc powder contained in a 100 mL round flask: a mixture of
1.3 g of zinc powder, 0.1 g of mercury(II) chloride, 0.06 mL of
concentrated hydrochloric acid, and 1.5 mL of water was stirred for 5
min. The liquid was decanted as completely as possible. Then 0.8 mL
of water, 2 mL of concentrated HCl, 2 mL of pure toluene, and 0.255
g (1 mmol) of 4-(4-biphenyl)-4-oxobutanoic acid were added consecu-
tively. The flask was fitted with a reflux condenser connected to a gas
absorption trap, and the reaction mixture was boiled vigorously for 30
h. During the refluxing period, three 0.4 mL portions of concentrated
HCl were added at approximately 6 h intervals to maintain the
concentration of the acid. The mixture was allowed to cool to room
temperature to separate into two layers. The aqueous portion was diluted
with 10 mL of H2O and extracted several times with ether. The
combined extracts were dried over MgSO4, and the solvent was removed
under reduced pressure to give the crude product, which was purified
by silica column chromatography (ethyl acetate/n-hexane 1:1 as eluent)
to give 4-(4-biphenyl)butanoic acid, a white needle solid, with 83%
Figure 1. 31P spectra for mixtures of DBBPC/DHPC (10% in D2O) with
different mole ratios at 25 °C.
chromatography (65:35:4 CHCl3/CH3OH/H2O) to give OBPC (48%
yield): 1H NMR (400 MHz, CDCl3): δ 7.8 (m, 8H, Ar), 7.1 (m, 8H,
Ar), 5.54 (m, 1H, CH), 4.52 (dd, 1H, CH), 4.2 (m, 2H, POCH2CH2N),
4.1 (dd, 1H, CH), 3.9 (m, 2H, CH2OCO), 3.7 (m, 2H, CH2N), 3.2 (s,
9H, N(CH3) ), 2.6 (t, 2H, CH2), 1.5 (m, 2H, -CH2-), 1.23 (s, 12H,
3
1
fatty CH2), 0.85 (t, 3H, -CH3).
yield. H NMR (400 MHz, CDCl3): δ 7.6 (d, 2H, Ar), 7.55 (d, 2H,
To prepare samples for NMR studies, 0.035 g of DBBPC was put
in an NMR tube, and 0.4 mL of 0.1 M NaCl in D2O was added. A
piece of Teflon tape was placed on top of the tube, which was then
capped tightly. Then, the NMR tube was subjected to repeated cycles
of heating, vortexing, and back-and-forth centrifugation until the solid
dissolved. A calculated amount of DHPC solution (30 wt % in D2O)
was then added into the NMR tube, and several cycles of heating,
vortexing, and centrifugation were repeated until the solution became
homogeneous. For the comparison of flipped bicelles with the common
bicelles, a sample of a 2.5:1 mixture of DLPC/DHPC in a solution of
0.1 M NaCl in D2O was prepared as well. 31P (162 MHz), 23Na (106
MHz), and 2H (61.4 MHz) spectra were acquired using a Varian
UNITY/INOVA 400 spectrometer at 9.4 T, usually with 100 scans.
Ar), 7.45 (m, 2H, Ar), 7.36 (m, 1H, Ar), 7.28 (d, 2H, Ar), 2.7 (m, 2H,
CH2), 2.1 (m, 2H, CH2).
1-Dodecanoyl-2-(4-(4-biphenyl)butanoyl)-sn-glycero-3-phospho-
choline (DBBPC, 4).16,18 1-Lauroyl-2-hydroxy-sn-glycero-3-phospho-
choline (0.22 g, 0.501 mmol) and 4-(4-biphenyl)butanoic acid (0.32 g,
1.328 mmol) were dissolved in CH2Cl2 (7 mL). 4-(Dimethylamino)-
pyridine (DMAP) (0.50 mmol) and a solution of 1,3-dicyclohexylcar-
bodiimide (DCC) (1.34 mmol) in CH2Cl2 (2 mL) were added, and the
solution was stirred at room temperature for 2 days. A white precipitate
was removed by filtration through a small cotton wool plug in a pipet.
The product was obtained by rotary evaporation of the filtrate and
purified twice by column chromatography (65:35:4 CHCl3/CH3OH/
H2O) to give DBBPC (60% yield): 1H NMR (400 MHz, CDCl3): δ
7.57 (d, 2H, Ar), 7.5 (d, 2H, Ar), 7.4 (m, 2H, Ar), 7.3 (m, 1H, Ar), 7.2
(d, 2H, Ar), 5.15 (m, 1H, CH), 4.25 (dd, 1H, CH), 4.2 (m, 2H, POCH2-
CH2N), 4.1 (dd, 1H, CH), 3.9 (m, 2H, CH2OCO), 3.7 (m, 2H, CH2N),
Results and Discussion
An initial attempt was made to formulate bicelles from
mixtures of DHPC and the synthetic aromatic PC OBPC (5),
which has two identical chains with phenyl rings. The reasoning
is that OBPC is more similar to DMPC and DLPC than DBPC
or DBBPC is, and the presence of two phenyl rings would also
result in ∆ø > 0 so that the bicellar normal would align parallel
to the magnetic field. Unfortunately, samples with a wide range
of composition did not form a homogeneous phase over the
temperature range of 20-75 °C. Therefore, subsequent effort
was concentrated on the study of DBBPC/DHPC systems. For
a mole ratio between 5.1:1 and 6.5:1, it was found that the
mixtures form stable bicelle solutions from 10 to 75 °C, while
the bicelles coexist with isotropic micelles above 54 °C. The
details are discussed in the following.
The 31P NMR spectra of several mixtures of DBBPC/DHPC
at 25 °C are shown in Figure 1. When the DBBPC/DHPC mole
ratio (q) was 7:1 and 5:1, the spectra are very broad, showing
typical partial powder patterns. This is an indication that the
lamellar bilayers of the PCs do not have macroscopic alignment
in the magnetic field. When q is between 6.5:1 and 5.1:1, the
31P spectra show two peaks at about 19 and 5 ppm, respectively.
3.3 (s, 9H, N(CH3) ), 2.6 (t, 2H, CH2), 2.3 (t, 2H, CH2), 2.2 (t, 2H,
CH2), 1.9 (t, 2H, CH2), 1.4 (m, 2H, -CH2-), 1.16 (s, 16H, fatty CH2),
0.8 (t, 3H, -CH3); mass spectrum (FAB): m/z 662.5 (calculated 661.8).
3
1,2-(4-Octylbenzoyl)-sn-glycero-3-phosphocholine (OBPC, 5).18,19
1-R-Glycerophosphocholine (1:1 cadmium chloride adduct) (200 mg,
0.447 mmol) and 4-octylbenzoic acid (425 mg, 1.816 mmol) were
dissolved in CH2Cl2 (6 mL). 4-(Dimethylamino)pyridine (DMAP)
(1.816 mmol) and a solution of 1,3-dicyclohexylcarbodiimide (DCC)
(1.816 mmol) in CH2Cl2 (2 mL) were added, and the solution was stirred
at room temperature for 4 days. Then the reaction mixture was cooled
to -5 °C, the precipitate was filtered off, and the filtrate was evaporated.
The residue was dissolved in a 4/5/1 chloroform/methanol/water
mixture, and the solution was slowly passed through a column packed
with a mixture of 15 mL of Amberlite IRC-50 and 15 mL of Amberlite
IR-96. An additional 50 mL of 4/5/1 chloroform/methanol/water mixture
was passed through the column, and the combined filtrate was
evaporated in vacuo. The crude product was purified twice by column
(18) Anikin, A.; Chupin, V.; Anikin, M.; Serebrennikova, G. Makromol. Chem.
1993, 194, 2663.
(19) (a) Huang, Y.; Picq, M.; Nemoz, G.; Doutheau, A.; Lagarde, M. Chin. J.
Appl. Chem. 1999, 16, 88. (b) Binder, H.; Anikin, A.; Lantzsch, G.; Klose,
G. J. Phys. Chem. 1999, 103, 461.
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A R T I C L E S
Tan et al.
Figure 3. Temperature dependence of the 31P chemical shifts of the major
peak (solid squares) and the minor peak (open squares), mole ratio of
DBBPC/DHPC in the bicelle fraction (solid circles), and the relative intensity
of isotropic PC micelles (diamonds). The lines are drawn to guide the eye
only.
Figure 2. 31P spectra for a 6:1 DBBPC/DHPC mixture (10% in D2O) at
different temperatures. The small peaks at ca. 0, 1, 7, and 10 ppm are due
to impurities present in the starting material.
Figure 4. Schematic representation of morphological changes of the
DBBPC/DHPC mixtures from low temperature to high temperature. The
DHPC molecules are denoted with a solid headgroup; the sizes of the bicelles
and micelles are not in proportion. Small amounts of monomers would also
be present, but they are not shown in the diagram.
The relative areas of the peaks are consistent with the corre-
sponding mole ratio of the two PCs. By comparing the spectral
pattern with that of common bicelles1a,2 and those calculated
from theoretical considerations,20 it is clear that the normal of
the bicelles formed by the DBBPC/DHPC mixtures aligns
parallel to the magnetic field. This situation is similar to that
for bicelles formulated by mixing DBPC (3) and DHPC.16
However, the DBPC/DHPC bicelles are stable only within a
narrow range of composition. The increased stability of the
DBBPC/DHPC bicelles over a larger range of q is likely due
to the presence of two more CH2 spacers in the chain containing
the biphenyl unit, making the chain more flexible.
The thermal stability of the DBBPC/DHPC bicelles with q
) 6 was studied by following the 31P spectra (Figure 2). The
two 31P peaks characteristic of the “flipped” bicelles indicate
that they are thermally stable over the temperature range (10-
75 °C). With increasing temperature up to about 54 °C, the
chemical shifts of the major and minor peaks change in opposite
directions (Figure 3, squares). Above 54 °C, the 31P chemical
shift of the major peak begins to increase with temperature. In
the meantime, a new peak near 0 ppm, which is characteristic
of isotropic PC micelles, starts to appear, and its intensity
increases with temperature at the expense of the peaks of the
bicelles (Figure 3, diamonds). The change of the spectra with
temperature is reversible. This indicates that there is an
equilibrium between bicelles and micelles above 54 °C, and a
schematic diagram of the temperature dependence of the
DBBPC/DHPC system is shown in Figure 4. Since the short-
chain DHPC molecules are more favorable to form aggregates
with larger curvatures, it is most likely that the isotropic micelles
are composed of mainly DHPC, as depicted in the schematic
diagram. Because of this, the ratio of DBBPC/DHPC in the
bicellar fraction increases with increasing temperature (Figure
3, solid circles), which would reduce the number of holes in
the perforated bilayers. This situation is quite different from
that of the DBPC/DHPC bicelle, for which there is a more
complicated equilibrium from 45 to 50 °C, but the bicelles
disappear above 55 °C, leaving only isotropic micelles.16 The
aliphatic DLPC/DHPC bicelles also have lower thermal stability,
and phase separate at about 50 °C.
A simulation of the 31P spectra of phospholipid bicelles
indicated that the areas under the peaks are dependent on the
mole ratio q, while the chemical shifts are not.20 When the
bicelle normal changes its alignment from perpendicular to
parallel without adding paramagnetic ions, the 31P peaks would
have twice the chemical shifts (with respect to the isotropic peak,
which is close to 0 ppm with respect to H3PO4) and opposite
signs.20 Although this simulation was made using the disklike
model, the argument would hold for the perforated bilayer model
as well. However, even though the observed 31P peaks of the
DBBPC/DHPC bicelles do have signs opposite those of DLPC/
DHPC bicelles, the shifts are not exactly twice (Figure 3). At
30 °C, the chemical shift difference between the major and
minor peaks in the DLPC/DHPC bicelles is -4.8 ppm, whereas
the corresponding difference for DBBPC/DHPC bicelles is 12.3
ppm, which is much more than a factor of -2 times the first
value. A similar result was observed in our previous studies in
DBPC/DHPC system,16 and it was suggested that the reason
for this is the different conformations of the polar headgroup
of the long-chain PC in the two types of bicelles. Obviously
(20) Picard, F.; Paquet, M.-J.; Levesque, J.; Be’langer, A.; Auger, M. Biophys.
J. 1999, 77, 888.
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Properties of Phospholipid Bicelles
A R T I C L E S
Figure 5. 2H and 23Na spectra of a 6:1 DBBPC/DHPC mixture (10% in
D2O) at 25 °C.
Figure 6. Temperature dependence of 2H (squares) and 23Na (circles)
quadrupole splitting. The open symbols are data for DLPC/DHPC bicelles
(mole ratio 2.5:1, 21% in D2O with 0.1 M NaCl), and the closed symbols
are data for DBBPC/DHPC bicelles (mole ratio 6:1, 10% in D2O with 0.1
M NaCl). The lines are drawn to guide the eye only.
the asymmetry in the two chains contributes to the conforma-
tional change of the polar headgroup. When the temperature
increases from 10 to 54 °C, the conformation of the headgroup
in DBBPC would change gradually. In the meantime, the order
parameter of the normals of the bicelles would decrease slightly
due to increasing thermal motions of the bilayer.
temperature dependence of the orientational distribution of the
bound water molecules with respect to the direction of the lipid
aggregate.22 For the DLPC/DHPC bicelles above ca. 50 °C, an
isotropic 2H peak appears in the middle of the doublet because
the system undergoes phase separation. As a consequence, the
2H quadrupole splitting in the liquid crystalline phase shows a
Both of these factors would contribute to the decrease in the
31P chemical shift of the major peak, which is due to the DBBPC
molecules, with increasing temperature below 54 °C (Figure 3,
solid squares). The minor peak is due to the DHPC molecules
which mostly occupy the rims of the holes. They are expected
to be less affected by the decrease in the order parameter of the
bicelle normal, and the change in their headgroup conformation
with temperature is expected to be much less due to the
symmetrical nature of the two chains in DHPC. Consequently,
the 31P chemical shift of the minor peak increases with
temperature, but much more slowly (Figure 3, open squares).
Above 54 °C, the conformation of the DBBPC headgroup is
expected to continue its gradual change, but the reduction in
the number of holes (Figure 4) would make the bilayers slightly
less flexible and raise the order parameter of the normal.
Therefore, the 31P chemical shift of the major peak starts to
increase; the minor peak is less affected, but the slope of the
temperature dependence does show a change at about 54 °C
(Figure 3). Because of the complexity of the two contributions,
we have not attempted a quantitative analysis of the 31P chemical
shift data.
2
sudden change in the slope (Figure 6). In comparison, the H
doublet splitting for the DBBPC/DHPC bicelles is smaller, but
it changes smoothly with temperature, showing no isotropic
peak. The temperature dependence of the 2H quadrupole splitting
(Figure 6) mirrors that of the major 31P peak. This indicates
that the orientational ordering of the D2O molecules is mainly
determined by those bound to the headgroups of DBBPC, which
is perfectly reasonable.
For the 23Na NMR of the Na+ ions, quadrupole splitting were
observed for the DLPC/DHPC bicelles as well as for the
“flipped” DBPC/DHPC and DBBPC/DHPC bicelles (Figure 5B;
in our previous work it was erroneously stated that the DLPC/
DHPC bicellar system showed no 23Na quadrupole splitting16).
The presence of 23Na splitting is due to an induced asymmetry
in the hydration sheath of the cations on the bilayer surface,
which are in rapid exchange with the Na+ ions in the bulk.23
2
Similar to the H splitting, the temperature dependence of the
To further investigate the behavior of the DBBPC/DHPC
bicelles, we studied the 2H NMR of D2O and the 23Na NMR of
NaCl in the mixtures to obtain complementary information.
In most aqueous lyotropic liquid crystals, 2H quadrupole
splitting can be observed for D2O. If the mesophase is
macroscopically aligned in the magnetic field, the 2H spectrum
shows a doublet; if there is no macroscopic alignment, the
spectrum exhibits a partial powder pattern. The 2H spectrum of
D2O in the DBBPC/DHPC mixture does show two distinct peaks
(Figure 5A), consistent with the presence of macroscopic
alignment of the bicelles. For most thermotropic liquid crystals
23Na quadrupole coupling for the DLPC/DHPC bicelles shows
a break at ca. 50 °C (Figure 6) due to phase separation. The
isotropic 23Na peak coincides with the central peak of the signal
in the micellar phase, and the peak intensity increases rapidly
with the increase in temperature. On the other hand, the 23Na
quadrupole splitting for the DBBPC/DHPC bicellar system
increases smoothly with temperature (Figure 6) since there is
no phase separation. Because of the presence of the hydration
sheath, the Na+ ions cannot approach the polar headgroups of
the phospholipids as closely as the water molecules. Therefore,
the morphological change in the system above 54 °C has a
2
and many lytropic liquid crystals, the H quadrupole splitting
2
smaller effect on the 23Na quadrupole splitting than the H
decreases with increasing temperature because of the decrease
in the order parameter. However, the opposite trend has been
observed for some lytropic liquid crystals21 because of the
quadrupole splitting (Figure 6).
(22) Mantsch, H. H.; Saito, H.; Smith, I. C. P. Prog. Nucl. Magn. Reson.
Spectrosc. 1997, 11, 211.
(21) (a) Hakala, M. R.; Wong, T. C. Langmuir 1986, 2, 83. (b) Guo, W.; Wong,
T. C. Langmuir 1987, 3, 537. (c) Rapp, A.; Ermolaev, K.; Fung, B. M. J.
Phys. Chem. B 1999, 103 (10), 1705.
(23) Soderman, O.; Arvidson, G.; Lindblom, G.; Fontell, K. J. Biochem. 1983,
134, 309.
(24) Benesi, A. J.; Brant, D. A. Macromolecules 1985, 18, 1109.
9
J. AM. CHEM. SOC. VOL. 124, NO. 39, 2002 11831

A R T I C L E S
Tan et al.
Table 1. Some 1H-13C Dipolar Coupling Constants (D, in Hz) of
in the table show that the signs of the 1H-13C dipolar coupling
constants of maltotriose in the DBBPC/DHPC bicellar solutions
are the same as those in DBPC/DHPC solutions and opposite
those in DLPC/DHPC bicelles at all three temperatures,
confirming the conclusion that the bicelle normal does align
parallel to the magnetic field. At 75 °C, there is an equilibrium
between bicelles and micelles (Figure 4), which would reduce
the amount of bilayers responsible for the alignment. However,
this is compensated for by the increase in the order parameter
30 mM Maltotriose in Three Bicellar Solutions in D2Oa
1â
1R
4′′
5R
2.6
-2.1
-1.0
-0.1
-0.3
2â
DLPC/DHPC bicellesb
DBPC/DHPCc
2.5
-2.7
-2.61
-2.31
-3.14
-4.0
10.2
7.4
8.3
7.6
-8.0
18.3
15.7
15.9
15.4
4.6
-7.9
-5.6
-6.0
-5.0
DBBPC/DHPC (25 °C)d
DBBPC/DHPC (50 °C)d
DBBPC/DHPC (75 °C)c
a The values were calculated from the splittings (∆) using the formular
∆ ) 2D + J, where J’s are scalar coupling constants determined from a
solution in pure D2O.24 1â, 1R, 4′′, 5R, and 2â are peak assignments.
b DLPC/DHPC (3/1, 10% w/v, 30 °C). c DBPC/DHPC (6/1, 11% w/v, 25
°C).16 d DBBPC/DHPC (6/1, 10% w/v).
1
of the bilayer normal, so that the H-13C dipolar coupling
constants are comparable to those at 25 °C.
In conclusion, it has been shown that the DBBPC/DHPC
bicelles align with their normals parallel to the magnetic field.
Because they are stable in larger composition and temperature
ranges compared with the DBPC/DHPC bicelles, they are a
better complementary system to common PC bicelles. The better
thermal stability of these “flipped” bicelles would also be
advantageous for the study of protein folding or thermophilic
proteins.
2
It is to be noted that both the H and the 23Na quadrupole
splittings for the DBBPC/DHPC solution are smaller than those
for the DLPC/DHPC solution. The reasons for this would
include different lipid concentrations and differences in the
alignment of the bicelle normal and the headgroup conforma-
tions in the two systems, but estimations of the contribution of
each factor are difficult to make.
To test the application of the new DBBPC/DHPC bicellar
system as an ordering medium for biological molecules, HMQC
(heteronuclear multiple quantum correlation) NMR experiments
were performed for a trisaccharide, maltotriose, in a DBBPC/
DHPC bicellar solution. The results for the best resolved 13C
peaks are listed in Table 1, and those for the DBPC/DHPC and
DLPC/DHPC systems are also listed for comparison. The data
Acknowledgment. This research was supported by the
Oklahoma Center for the Advancement of Science and Technol-
ogy under Grant HR-98080 and the National Science Foundation
under Grant DMR-0090218.
JA027079N
9
11832 J. AM. CHEM. SOC. VOL. 124, NO. 39, 2002