From the
Apolipophorin-III (apoLp-III) from Manduca sexta can
exist in two alternate states: as a globular, lipid-free helix bundle
or a lipid surface-associated apolipoprotein. Previous papers (Ryan R.
O., Oikawa K., and Kay C. M.(1993) J. Biol. Chem. 268,
1525-1530; Wientzek M., Kay C. M., Oikawa K., and Ryan R.
O.(1994) J. Biol. Chem. 269, 4605-4612) have
investigated the structures and properties of apolipophorin-III from
M. sexta in the lipid-free state and associated to lipids.
Association of apoLp-III with dimyristoylphosphatidylcholine vesicles
leads to the formation of uniform lipid discs with an average diameter
and thickness of 18.5 ± 2.0 and 4.8 ± 0.8 nm,
respectively. These discs contain six molecules of apoLp-III.
Geometrical calculations based on these data, together with x-ray
crystallographic data from the homologous L. migratoria apoLp-III (Breiter D. R., Kanost M. R., Benning M. M., Wesenberg
G., Law J. H., Wells M. A., Rayment I., and Holden H. M.(1991)
Biochemistry 30, 603-608), have allowed the presentation
of a model of lipid-protein interaction, in which the
Apolipophorin-III (apoLp-III)
To provide experimental
evidence of the validity of this model, Fourier transform
infrared-attenuated total reflection (ATR) spectroscopy was used here
to gain information about the secondary structure and orientation of
apoLp-III with regard to the lipid bilayer. This technique has been
applied successfully to deal with membrane structural problems in our
laboratory (Goormaghtigh et al., 1990, 1991a, 1991b, 1994a,
1994b; Challou et al., 1994; Sonveaux et al., 1994,
Vandenbussche et al., 1992). The results on the secondary
structure of the protein in the apoLp-III
Fig. 1
, A and B, and 2, A and B, show the IR spectra of a pure DMPC preparation and
of the apoLp-III
The amide I region of
the spectra (1700-1600 cm
The
orientation of the lipid acyl chains was also assessed. In DMPC, the
hydrocarbon chain in the
These results experimentally confirm the previously proposed model
of discoidal DMPC particles containing
A significant shift of the amide I peak from 1653 to 1646
cm
We thank R. Bradley and D. G. Scabra for assistance
with electron microscopy and H. de Jongh for assistance with CD
spectrometry.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-helices of
the apoLp-III orient perpendicular to the phospholipid chains and
surround the lipid disc. Here, using polarized Fourier
transform-attenuated total reflection infrared spectroscopy, we provide
the first experimental evidence of a unique perpendicular orientation
of the
-helices with respect to the fatty acyl chains of the
phospholipids in the disc.
(
)
is an
abundant hemolymph protein in the sphynx moth, Manduca sexta,
that normally exists in a lipid-free state (Shapiro and Law, 1983).
ApoLp-III is a single chain polypeptide of 166 amino acids
(M
= 18,300), which exists as a monomer in
solution and is rich in
-helix structure (Cole et al.,
1987; Kawooya et al., 1986). The three-dimensional structure
of lipid-free apoLp-III from Locusta migratoria has been
elucidated by x-ray crystallography (Holden et al., 1988;
Breiter et al., 1991). L. migratoria apoLp-III
contains five long amphipathic
-helices in a bundle connected by
short loops. There is an approximate 50% homology between M. sexta and L. migratoria apoLp-III amino acid sequences, and
these apoLp-IIIs have been found to be functionally indistinguishable
(Van der Horst et al., 1988). The two apoLp-IIIs likely
possess similar amphipathic helix folding motives (Wientzek et
al., 1994). When presented with a lipid surface, it was suggested
that apoLp-III undergoes a conformational change that exposes the
hydrophobic faces of the helices, thereby permitting interaction with
lipid surfaces. Association of apoLp-III with DMPC vesicles results in
the formation of uniform discs with an average diameter and width of
18.5 ± 2 nm and 4.8 ± 0.8 nm, respectively. Calculations
based on the experimental characterization of these complexes together
with the x-ray crystallographic data from L. migratoria apoLp-III allowed the presentation of a model of lipid-protein
interaction (Wientzek et al., 1994). In this model,
apolipoprotein helices are oriented perpendicular to the lipid acyl
chains and surround the phospholipid disc.
DMPC complexes are in
good agreement with the previous CD measurements (Ryan et al.,
1993). About the orientation, we provide the first experimental
evidence of a unique orientation of the apoLp-III helical domains
perpendicular to the lipid acyl chains. This topology is quite opposite
to that described for the apoA1
DMPC (Brasseur et al.,
1990) and apoA1
dipalmitoylphosphatidylcholine complexes (Hefele
Wald et al., 1990), where the amphipathic helices were
oriented parallel to the acyl chains.
Materials
Dimyristoylphosphatidylcholine
was purchased from Sigma. Phospholipid (choline) enzymatic colorimetric
analysis kit was obtained from Wako Pure Chemical Industries, Ltd.
(Osaka, Japan). Deuterium oxide was from Janssen Chimica (Geel,
Belgium).
Purification of Apolipophorin
III
apoLp-III was isolated and purified from hemolymph of
adult M. sexta with some modifications of the method by Wells
et al.(1985). The purity of the apoLp-III was evaluated by
SDS-polyacrylamide gel electrophoresis, and the pure apoLp-III was
stored lyophilized at -20 °C.
Preparation of DMPC Vesicles
DMPC
vesicles were prepared by modification of the method of Swaney(1980).
DMPC was dissolved in chloroform:methanol mixture (3:1), evaporated to
dryness as a thin film under an inert atmosphere, and dried further for
2 h, followed by the addition of 10 mM Tris-HCl, pH 7.5. The
mixture was warmed to 50 °C, vortexed, and then sonicated in a bath
sonicator for about 20 min.
Preparation and Isolation of DMPC
The preparation and isolation of
DMPCapoLp-III
Complexes
apoLp-III complexes were performed as described by
Swaney(1980) and Rifici et al.(1985) with some modifications.
ApoLp-III was dissolved in 10 mM Tris-HCl, pH 7.5, and added
to the DMPC vesicles at a lipid:protein ratio of 2.5:1 (w/w) (molar
ratio of about 70:1). After a brief mixing in a vortexer, the mixture
was incubated at 24 °C for 18 h. The density of the sample was
adjusted to 1.21 g/ml with KBr in a final volume of 2.5 ml and placed
in a 5-ml Quick-Seal tube, layered with 0.9% saline, and centrifuged at
65,000 rpm for 3 h at 4 °C. At the end of the centrifugation,
fractions of 0.5 ml were removed from the top of the tube and analyzed
for protein and phospholipid content. The DMPC
apoLp-III complexes
were localized by the coenrichment of protein and phospholipid, pooled,
and dialyzed extensively against 10 mM Tris-HCl, pH 7.5. The
protein content of the final DMPC
apoLp-III complexes was
evaluated by amino acid analysis (Beckman System 6300 Amino Acid
Analyzer, System Gold Version 6/01) and the phospholipid content by
colorimetric kit for choline. The complex had a lipid:protein weight
ratio of 7.1:1 (molar ratio of 190:1). Control DMPC alone and
apoLp-III
DMPC complexes were adsorbed to carbon-coated grids,
stained with 2% sodium phosphotungstate, and viewed under a Philips FM
420 electron microscope operated at 100 kV (Wientzek et al.,
1994). In addition to apoLp-III complexes, DMPC vesicles without added
apoLp-III were employed in parallel control experiments.
IR Spectroscopy
Spectra were recorded on
a Perkin-Elmer infrared spectrophotometer 1720X equipped with a
Perkin-Elmer microspecular reflectance accessory and a polarizer mount
assembly with a gold wire grid element. The internal reflection element
was a germanium ATR plate (50 20
2 mm, Harrick EJ2121)
with an aperture angle of 45° yielding 25 internal reflections. 128
accumulations were performed to improve the signal/noise ratio. The
spectrophotometer was continuously purged with air dried on a silicagel
column (5
130 cm) at a flow rate of 7 liters/min. Spectra were
recorded at a nominal resolution of 2 cm
. At the end
of the scan, they were transferred from the memory of the
spectrophotometer to a computer for subsequent treatments. All the
measurements were made at 20 °C.
Samples Preparation
Oriented multilayers
were formed by slow evaporation of 100 µl of the sample on one side
of the ATR plate. The ATR plate was then sealed in a universal sample
holder (Perkin-Elmer 186-0354) and hydrated by flushing
DO-saturated N
(room temperature) for 3 h.
Secondary Structure Determination
Fourier
self-deconvolution was applied to increase the resolution of the
spectra in the amide I` region, which is the most sensitive to the
secondary structure of proteins. The self-deconvolution was carried out
using a Lorentzian line shape for the deconvolution and a Gaussian line
shape for the apodization. To quantify the area of the different
components of amide I` revealed by the self-deconvolution, a least
square iterative curve fitting was performed to fit Lorentzian line
shapes to the spectrum between 1700 and 1600 cm.
Prior to curve fitting, a straight base line passing through the
ordinates at 1700 and 1600 cm
was subtracted. The
spectrum arising from the lipid part of the system was found to be
completely flat between 1700 and 1600 cm
and was
therefore not subtracted. To avoid introducing artifacts due to the
self-deconvolution procedure, the fitting was performed on the
non-deconvolved spectrum. The proportion of a particular structure is
computed to be the sum of the area of all the fitted Lorentzian bands
having their maximum in the frequency region where that structure
occurs divided by the area of all the Lorentzian bands having their
maximum between 1689 and 1615 cm
(Goormaghtigh
et al., 1990).
Orientation of the Secondary
Structures
For the amount of material used throughout this
work, film thickness remains small when compared to the IR wavelength.
This allows the ``thin film'' approximation to be used for
the establishment of the equations describing the dichroic ratio as a
function of the orientational order parameter (Goormaghtigh and
Ruysschaert, 1990). The potential energy distribution of amide I
consists in about 80% (C=O), 10%
(C-N), and 10%
(N-H). In an
-helix, the main transition dipole moment
lies approximately parallel to the helical axis, while in an
anti-parallel
-sheet, the polarization is predominantly
perpendicular to the fiber axis. It is therefore possible to determine
the mean orientation of the
-helix and
-sheet structures from
the orientation of the peptide bond C=O group (Goormaghtigh and
Ruysschaert, 1990). When this information was desired, additional
spectra were recorded with parallel (0°) and perpendicular
(90°) polarized incident light with respect to a normal to the ATR
plate. Polarization was expressed as the dichroic ratio
R
=
A
/A
. The mean angle between the
helix axes and a normal to the ATR plate surface was then calculated
from R
. In these calculations, a 27° angle
between the long axis of the
-helix and the C=O dipole
moment was considered (Goormaghtigh and Ruysschaert, 1990; Goormaghtigh
et al., 1994a). The
(CH
)
transition at 1200 cm
, whose dipole lies parallel to
the all-trans hydrocarbon chains, was used to characterize the
lipid acyl chain orientation (Fringeli and Günthard, 1981).
CD Spectroscopy
Spectra were recorded
between 260 and 190 nm with a resolution of 0.5 nm on a Jasco J-710
spectropolarimeter controlled by Jasco software. The spectropolarimeter
was continuously purged with N. The cell used was a
cylindral quartz cell with a pathlength of 1 mm. The spectra were the
result of four accumulations at a scan speed of 50 nm/min with a
response time of 0.125 s. The concentration of the samples was 0.15 mg
of protein/ml.
DMPC complex (190:1 lipid:protein molar ratio),
respectively. The amide I region of the spectrum (1700-1600
cm
) (Fig. 2, A and B)
reveals a symmetric peak with a maximum at 1653 cm
characteristic of a mainly helical structure in good agreement
with the CD (Ryan et al., 1993) and x-ray (Breiter et
al., 1991) data. Since the CD spectra of the apoLp-III
DMPC
complexes provided evidence that apoLp-III becomes essentially
completely helical after interaction with DMPC (Ryan et al.,
1993), we assigned the 1653 cm
band to the
-helix structure. After exposure of the sample to
D
O-saturated N
for 10 h, the amide I` region
shifted from 1653 to 1646 cm
(Fig. 3, A and B). Compilation of literature data on the behavior of
the
-helix amide I component (Goormaghtigh et al., 1994a)
reveals that a 1646-cm
frequency is well within the
range of frequencies characteristic of deuterated helices. To make sure
that the shift of the amide I maximum from 1653 to 1646 cm
we observed after deuteration was not due to a change in the
protein secondary structure upon incubation in the presence of
D
O, we exchanged the aqueous buffer of a similar sample by
either D
O or H
O by dialysis and recorded CD
spectra. It turned out that this CD spectrum of the protein in
D
O was exactly superimposable to the CD spectrum of the
sample dialyzed against H
O (data not shown). We can
therefore assign unambiguously the shift of the amide I to the
deuteration of the
-helices of the apoLp-III.
Figure 1:
IR spectra of a DMPC film. SpectrumA was obtained with 90° polarized light and
spectrumB with 0° polarized light. TraceC represents the difference between spectrumA and spectrumB. A positive deviation
in spectrumC from a horizontal base line indicates a
higher absorbance at 90° than at 0° and reflects a preferential
perpendicular orientation of the dipole transition, relative to the
germanium surface. A negative deviation in spectrumC indicates that the dipole is preferentially oriented parallel to
the germanium surface. The optical density amplitudes of spectraA-C are 0.255, 0.211, and 0.128, respectively. The
sample has been deuterated as described under ``Experimental
Procedures.''
Figure 2:
IR spectra of the apoLp-IIIDMPC
complex. SpectrumA was obtained with 90°
polarized light and spectrumB with 0° polarized
light. TraceC represents the difference between
spectrumA and spectrumB. The
optical density amplitudes of spectraA-C are
0.239, 0.141, and 0.30, respectively.
Figure 3:
IR spectra of the deuterated
apoLp-IIIDMPC complex. SpectrumA was obtained
with 90° polarized light and spectrumB with
0° polarized light. TraceC represents the
difference between spectrumA and spectrumB. The optical density amplitudes of spectraA-C are 0.255, 0.152, and 0.30, respectively. The
sample has been deuterated as described under ``Experimental
Procedures.''
Components of the
amide I` revealed by Fourier self-deconvolution were quantified by a
least square iterative curve fitting as described (Goormaghtigh et
al., 1990). The calculated percentage of helical secondary
structure (70-80%) (data not shown) is in good agreement with the
Provencher-Glöckner analysis of the CD spectra, revealing a
completely -helical structure for the apoLp-III upon complex
formation (Wientzek et al., 1994).
) was used to
determine the orientation of the
-helices in apoLp-III relative to
the DMPC hydrocarbon chains of the bilayers in the apoLp-III
DMPC
complex. A dichroic spectrum, obtained by subtracting the spectrum
recorded with 0° polarized light from the spectrum recorded with
90° polarized light, has a positive deviation when the dipole
moment was oriented perpendicular to the germanium crystal plane and a
negative deviation when the dipole moment was oriented parallel to the
germanium plane (Goormaghtigh and Ruysschaert, 1990). Here, for both
the undeuterated (Fig. 2C) and deuterated
(Fig. 3C) samples, the amide I band is strongly 0°
polarized, as shown by a negative deviation located in the helix region
of the amide I band, indicating an orientation of the
-helices
parallel to the germanium plate, i.e. parallel to the lipid
bilayer and perpendicular to the acyl chains. This orientation of the
helices is confirmed by the positive deviation in the amide II band at
1549 cm
corresponding to the
-helix amide II
band (Goormaghtigh et al., 1994a). Indeed, the amide I and
amide II polarizations are perpendicular (Krimm and Bandekar, 1986;
Tsuboi, 1962). A more quantitative estimation of the helix orientation
can be obtained after iterative least square curve fitting applied to
the deconvolved polarized spectra, which allows the computation of the
dichroic ratio for the
-helix components in the
apoLp-III
DMPC complex. While the isotropic dichroic ratio
(i.e. the dichroic ratio measured for a dipole with an
isotrope distribution) was 1.51, it was 1.21 for the amide I component,
indicating that the helix axis makes a maximum tilt with the germanium
surface of 20°. This value is a maximum estimate since taking into
account additional sources of disorder, e.g. a mosaic spread
(Rothschild and Clark, 1979), would further reduce this tilt.
-position in the gel state is
all-trans from the ester group to the methyl group. This
conformation allows a resonance to occur between the ester group and
the CH
groups of the chain, giving rise to the so-called
(CH
) progression between 1200 and 1350
cm
(peaks at 1201, 1230, 1257, 1280, 1304, and 1328
cm
) (Fig. 1-3). The peak at 1200
cm
was chosen to characterize the lipid acyl chain
orientation (Fringeli and Günthard, 1981). The strong 90°
polarization of this absorption peak (Fig. 1-3) indicates
that the all-trans hydrocarbon chains of DMPC are oriented
nearly normal to the germanium surface. The measured dichroic ratio is
3.4-4.0. Accordingly, the maximum tilt between acyl chain and a
normal to the germanium surface is equal to 20°. Additional source
of disorder would reduce this tilt. These data indicate that the lipid
hydrocarbon chains and the
-helices of the protein are oriented
almost perpendicular to each other in the apoLp-III
DMPC complex.
-helical segments of
apoLp-III organized perpendicular to the phospholipid acyl chains on
the periphery of the discoidal structure. It is to the best of our
knowledge the first experimental evidence of a protein interacting with
the lipid core and orienting normal with respect to the acyl chains.
was associated to the deuteration of the
apoLp-III helical domains. On the opposite, such a shift has not been
described after deuteration of the
apoA1
dipalmitoylphosphatidylcholine discoidal complex (Hefele
Wald et al., 1990) and is not observed for membrane and
soluble
-helix-rich proteins tested so far in our laboratory. This
raises the question of the helix structure (
,
angles,
hydrogen bonding). However, the frequency of the undeuterated form of
the protein is in good agreement with a large set of data collected in
the literature as reported by Goormaghtigh et al. (1994a).
This demonstrates that the geometry and hydrogen bonding in the helices
is close to this of a standard
-helix since the amide I frequency
is very sensitive to these parameters. CD data confirm on the other
hand the large amount of helix structure. We suggest that the peculiar
distribution of the hydrophilic and hydrophobic amino acids on the two
sides of the apoLp-III helices and the interaction with the lipid
bilayer is responsible for the amplitude of the shift. The high extent
of apoLp-III helices deuteration could be related to its interaction
with the lipid bilayer. The apoLp-III helices are large and normal with
respect to the acyl chains. The length of apoLp-III helices and the
contours of the lipid discs could give to the helices a curved shape,
which would increase the accessibility of their amide hydrogen,
involved in hydrogen bonds, to the solvent and favor the
hydrogen/deuterium exchange. Hydrogen/deuterium exchange has been shown
to be much more rapid in amphipathic helices distorted to a curved
structure than in non-distorted helices (Zhou et al., 1992).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.