Bolalipid membrane structure revealed by solid-state 2H NMR spectroscopy

Article abstract of DOI:10.1021/ja710190p

Membranes made from three specifically deuterium-labeled ether-linked bolalipids, [1′,1′,20′,20′-²H₄]C₂₀BAS-PC, [2′,2′,19′,19′-²H₄]C₂₀BAS-PC, or [10′,11′-²H₂]C₂₀BAS-PC, were analyzed by ²H NMR spectroscopy. Unlike more common monopolar, ester-linked phospholipids, C₂₀BAS-PC exhibits a high degree of orientational order throughout the membrane and the sn-1 chain of the lipid initially penetrates the bilayer at an orientation different from that of the bilayer normal, resulting in inequivalent deuterium atoms at the C1 position. The approximate hydrophobic layer thickness and area per lipid are 18.4 A and 60.4 A², respectively, at 25 °C, and their respective thermal expansion coefficients are within 20% of the monopolar phospholipid, DLPC. Copyright

Full text of DOI:10.1021/ja710190p

Published on Web 03/19/2008
2
Bolalipid Membrane Structure Revealed by Solid-State H NMR Spectroscopy
David P. Holland,† Andrey V. Struts,‡ Michael F. Brown,‡ and David H. Thompson*,†
Department of Chemistry, Purdue UniVersity, West Lafayette, Indiana 47907 and Department of Chemistry,
UniVersity of Arizona, Tucson, Arizona 85721
Received November 9, 2007; E-mail: davethom@purdue.edu
The development of membrane-protein based biotechnologies
necessitates the identification and design of new membrane
materials with enhanced stability. Bolalipids are an interesting class
of bipolar lipids that have been proposed for these applications.
Archaea are unicellular organisms capable of thriving under extreme
conditions, such as high temperature, high salt concentration, or
low pH. Archaea often possess significant amounts of bolalipids
that contain a hydrolysis resistant ether-linkage, as opposed to the
more labile ester-linked monopolar phospholipids of Eukaryota and
Bacteria. One distinctive feature of bolalipids is the presence of
membrane spanning alkyl chains, which are believed to be
responsible for their enhanced physical stability.1 This stability has
been utilized to develop planar supported membranes,2 to recon-
stitute and study integral membrane proteins,3 and as gene and
vaccine delivery vehicles.4 Most of these efforts have focused on
the use of synthetic archaeal lipid analogues due to the difficulties
of obtaining naturally occurring bolalipids in pure form.5,6 Given
the growing importance of these analogues, a better understanding
of their structural and dynamical properties is needed.
2
Solid-state H NMR spectroscopy is commonly used to probe
membrane structure and dynamics.7 2H NMR spectroscopy can
probe individual C-2H bonds in a membrane lipid or protein,
providing atomically resolved information on the local environment
of the deuteron. The observable quadrupolar couplings arise from
2
the interaction of the quadrupole moment of the H nucleus with
the electric field gradient of the C-2H bond. These couplings can
Figure 1. (A) Structure of non-deuterated C20BAS-PC. (B) Powder-type
be used to calculate segmental order parameters (SCD), which
(red) and de-Paked (blue) H NMR spectra of [1′,1′,20′,20′-2H4]C20BAS-
2
provide insight into the local membrane organization at the position
PC (1), [2′,2′,19′,19′-2H4]C20BAS-PC (2), and [10′,11′-2H2]C20BAS-PC (3)
of the C-2H bond. Here we utilized H NMR spectroscopy to
analyze three deuterium-labeled variants of a previously synthesized
bolalipid analogue, C20BAS-PC.6 The three compounds, [1′,1′,20′,-
20′-2H4]C20BAS-PC (1), [2′,2′,19′,19′-2H4]C20BAS-PC (2), and
[10′,11′-2H2]C20BAS-PC (3) (Figure 1), were synthesized with
deuterium labels on the sn-1 chain (Supporting Information).
Residual quadrupolar couplings were calculated from the de-Paked
2H NMR spectra.8 These splittings were used to calculate SCD, the
approximate hydrophobic layer thickness for half of the membrane
(Dc), and the area per lipid A using a first-order mean-torque
model.9 The data were compared to a monopolar lipid, dilauryl
phosphatidylcholine (DLPC).9
2
at 25 °C. (C) Powder-type 2H NMR spectra of [10′,11′-2H2]C20BAS-PC at
-60, 10, 15, 35, and 55 °C.
glycero-3-phosphocholine (DPPC) at the sn-2 C2 position10 and
the C1 position of both the sn-1 and sn-2 chains in the ether-linked
monopolar phospholipid, 1,2-dihexadecyl-sn-glycero-3-phospho-
ethanolamine (DHPE).11 It is believed that the inequivalence of
the two deuterium atoms arises from different glycerol H-C1-
O-C1′-2H alkyl dihedral orientations of the alkyl chain near the
bilayer interface.10
The segmental order parameters of the bolalipid can be used to
determine the conformation of the lipid within the membrane. We
were specifically interested in whether the bolalipids adopt a
transmembrane conformation, with the two polar head groups on
opposite sides of the membrane, or a looping conformer, with the
polar head groups on the same side of the membrane. The
centermost C-2H bonds should exhibit drastically different orienta-
tions with respect to the membrane normal between the two
conformers. This difference will be observed as a change in the
magnitude of the quadrupolar splitting, where the transmembrane
conformer generates a much larger splitting than that of the looping
conformer.12 A previous study of ester-linked bolaamphiphiles
resulted in mostly transmembrane conformers with a small (<10%)
In the high-temperature, liquid-crystalline state, 2 and 3 (Figure
1) yielded single quadrupolar splittings due to the equivalence of
all deuterium atoms in both compounds, resulting in order
parameters of |SCD| ) 0.21 and 0.23 at 25 °C, respectively.
However, 1 (Figure 1) gave two distinct quadrupolar splittings,
indicating the existence of two distinct environments for deuterium
in this molecule. These two components were equal in intensity at
two different temperatures (25 and 45 °C) in the liquid-crystalline
phase. Similar findings have been reported for 1,2-dipalmitoyl-sn-
† Department of Chemistry, Purdue University.
‡ Department of Chemistry, University of Arizona.
2
percentage of looping conformers.13 The H NMR spectrum of 3,
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C O M M U N I C A T I O N S
values of Dc and A of C20BAS-PC at 30 °C are 9.1 Å and 60.5
Å2, respectively (Figure 3). While DLPC has a larger value of Dc
than C20BAS-PC (10.5 vs 9.1 Å), the area per lipid is quite similar
(62.6 vs 60.5 Å2). Taking into consideration that a DLPC bilayer
consists of 22 methylene segments as opposed to 20 in C20BAS-
PC, these results demonstrate that the additional mass from the
DLPC membrane contributes to a thicker hydrophobic layer while
having a much smaller impact on A . The isobaric thermal
expansion coefficients for Dc and A (R| and R ) are given by the
slopes of ln Dc and ln A as a function of temperature. We report
Figure 2. (A) De-Paked quadrupolar splittings (∆νQ) of [10′,11′-2H2]C20-
(i)
BAS-PC as a function of temperature. (B) Order parameters |S | of C20-
CD
R| and R for C20BAS-PC as -2.4 × 10-3 and 3.6 × 10-3 K-1
,
BAS-PC (blue) at 25 °C and DLPC (red) at 30 °C as a function of carbon
number.
respectively. These data are very similar to those for DLPC (R| )
-3.0 × 10-3 and R ) 4.1 × 10-3 K-1), despite the very different
molecular structures and segmental order profiles.
These results provide insight into the structural and functional
properties of bolalipids and may aid our understanding of their
interactions with associated proteins. We are currently utilizing the
2H NMR spectroscopy results reported here to investigate the
mixing behavior of bolalipid/monopolar binary membrane systems.
Acknowledgment. We thank Victor Constantino for assistance
in material preparation and Gabriel Longo, Avigdor Leftin, and
Igal Szleifer for helpful discussions. We also gratefully acknowledge
the support from the National Institutes of Health Grants CA112427
and EY12049.
Supporting Information Available: Synthesis and characterization
of [1′,1′,20′,20′-2H4]C20BAS-PC (1), [2′,2′,19′,19′-2H4]C20BAS-PC (2),
and [10′,11′-2H2]C20BAS-PC (3). This material is available free of
charge via the Internet at http://pubs.acs.org.
Figure 3. (A) Hydrophobic layer thickness (DC) for [2′,2′,19′,19′-2H4]C20-
BAS-PC (green) and DLPC (red) as a function of temperature. (B) Natural
logarithm of hydrophobic layer thickness, (C) area per lipid, and (D) natural
logarithm of area per lipid as a function of temperature.
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It has been established that Dc, A , and thermal expansion
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splittings (cf. Supporting Information).9 Our data show that the
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