From the Department of Pharmacology,
Department of Medicine, Infectious Diseases Section, and the
Johnson Foundation for Molecular Biophysics, University of Pennsylvania
School of Medicine, Philadelphia, Pennsylvania 19104-6084 and the
§ Department of Biochemistry and Molecular Genetics and
¶ Department of Physiological Optics, Center for Macromolecular
Crystallography, University of Alabama at Birmingham Medical Center,
Birmingham, Alabama 35294
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ABSTRACT |
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The two main competing models for the structure
of discoidal lipoprotein A-I complexes both presume that the protein
component is helical and situated around the perimeter of a lipid
bilayer disc. However, the more popular "picket fence" model
orients the protein helices perpendicular to the surface of the lipid
bilayer, while the alternative "belt" model orients them parallel
to the bilayer surface. To distinguish between these models, we have investigated the structure of human lipoprotein A-I using a novel form
of polarized internal reflection infrared spectroscopy that can
characterize the relative orientation of protein and lipid components
in the lipoprotein complexes under native conditions. Our results
verify lipid bilayer structure in the complexes and point unambiguously
to the belt model.
Human apolipoprotein A-I
(apohA-I)1 comprises 70% of
the total protein found in plasma high density lipoproteins. As a
lipoprotein complex, apohA-I is thought to mediate a variety of
functions, including (i) the binding and transport of plasma lipids,
(ii) activation of lecithin-cholesterol acyltransferase (1), (iii) stimulation of cholesterol efflux from peripheral tissues (2, 3), and
(iv) uptake by the liver via a human apoA-I-specific receptor (4, 5).
The later two processes have been called "reverse cholesterol
transport" and may account for the inverse relationship observed
between plasma high density lipoprotein concentration and the risk of
developing coronary artery disease (6-8).
Sequence analysis suggested that apohA-I forms a series of amphipathic
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices (9), and this has been confirmed in the crystal structure
of a lipid-free amino-terminal truncation mutant of human apoA-I
(apo
(1-43)A-I) (10). However, it is not clear which of two
disparate models best describes the orientation of these helices within
the lipoprotein complex. One model positions the helices of apohA-I
around the perimeter of a lipid bilayer disc and orients them
perpendicular to the plane of the bilayer. This "picket fence"
model (Fig. 1, right) (11, 12)
is supported primarily by an internal reflection infrared spectroscopy
study of dried lipoprotein films (13). The "belt" model (Fig. 1,
left), on the other hand, also positions the helices of
apohA-I around the perimeter of a lipid bilayer disc, but orients them
parallel to the plane of the bilayer (14-16). The results of Borhani
et al. (10) and Rogers et al. (17, 18) are more
consistent with the belt model.
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Fig. 1.
Models of the lipoprotein complexes, and
schematic of the experimental design. A phospholipid monolayer was
applied to an octadecyltrichlorosilane-treated germanium crystal by
spreading 80 mol % DMPC and 20 mol % DMPS at the air-water interface
in a Langmuir trough, compressing this material to a surface pressure
of 38 dyne/cm, and placing the crystal flat onto the monolayer
(22-24). Polarized infrared light from a commercial FTIR spectrometer
was directed into the crystal, through a series of internal
reflections, and out to an external detector. When the lipoprotein
complexes in the subphase adsorbed onto the portion of the monolayer
that was in contact with the crystal, they attenuated the evanescent
field created by each internal reflection and produced an absorption
spectrum. Lipoprotein adsorption occurs to a significant extent only in
the presence of calcium, demonstrating that unadsorbed complexes do not
contribute to the spectroscopic signal and suggesting that calcium ions
form bridges between the seryl headgroups in the monolayer and in the
complexes. Helix orientations in the models are antiparallel, as
indicated by banded oval regions.
We have examined the orientation of protein and lipid components in
these lipoprotein complexes using specialized instrumentation for
performing polarized attenuated total internal reflection infrared
spectroscopy on supported lipid monolayers (19-24). In the presence of
1 mM Ca2+, lipoprotein complexes adsorb to
supported monolayers and permit one to record polarized internal
reflection infrared absorption spectra of oriented complexes (Fig. 1).
Since the complexes remain in buffer throughout the measurement, this
approach permits a direct spectroscopic study of uniformly oriented
lipoprotein complexes under native conditions (25).
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EXPERIMENTAL PROCEDURES |
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The 243-residue wild type apolipoprotein A-I (apohA-I) was
purified from human plasma, and a 200-residue NH2-terminal
deletion mutant (apo(1-43)A-I) was expressed and purified as
described previously (26-28). Lipid-free apoproteins were prepared for
study by dissolving them in 6 M guanidine hydrochloride, 20 mM HEPES, and 150 mM NaCl at pH 7.4, then
extensively dialyzed using tubing with a 5,000-dalton cutoff to remove
the guanidine. Following this, they were dialyzed against a
D2O-based buffer (30 mM HEPES, 150 mM NaCl, pD = 7.4). Protein concentrations were
determined in 6 M guanidine hydrochloride by absorption at
280 nm using extinction coefficients of 1.13 and 0.992 ml/(mg·cm) for
apohA-1 and apo
(1-43)A-1, respectively. Lipoprotein complexes were
reconstituted as discoidal lipoproteins using synthetic
dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylserine
(DMPS) from Avanti Polar Lipids, Inc. (Alabaster, AL). The lipid was a
mixture of DMPC:DMPS at a 9:1 molar ratio in organic solution, dried
with a stream of nitrogen, and then further dried under vacuum for at
least 4 h (15). The lipids were resuspended with proteins and 8 M guanidine hydrochloride in PBS buffer (0.02 M
sodium phosphate, 0.15 M sodium chloride, pH 7.4) and
diluted with PBS to a protein concentration of <1 mg/ml. This sample
was gently shaken at room temperature for 3-5 h and dialyzed
exhaustively against PBS with a 12-14-kDa molecular mass cutoff
membrane, followed by dialysis against 30 mM HEPES, pH 7.4, with a 50-kDa molecular mass cutoff membrane to remove excess free
protein. This procedure produced heterogeneously sized particles with
two to three discrete complexes between 10 and 13 nm in diameter as
confirmed by native gradient gel electrophoresis. Homogeneously sized
particles were produced by detergent dialysis (27), followed by gel
filtration to remove excess free protein. Prior to spectroscopic study,
the optically clear solution was dialyzed against 100 ml of 30 mM HEPES, pD 7.4, D2O buffer. This procedure
yielded homogeneously sized particles of approximately 7.8 nm in
diameter that migrated as a single band before and after prolonged
exposure to 1 mM Ca2+. The concentrations of
DMPS used in these experiments are well below the threshold at which
nonliquid lipid phases would be induced by 1 mM
Ca2+ (29).
Polarized attenuated total internal reflection Fourier-transform
(PATIR-FTIR) spectroscopy was performed on a previously
described instrument (19), modified such that the internal reflection crystal is applied flat onto a monolayer at the air-water
interface in a Langmuir trough (23). The angle of incidence between the IR beam and the crystal surface was 30°. The internal reflection crystal was treated with octadecyltrichlorosilane to render the surface
hydrophobic (19, 30). All spectra (1024 co-added interferograms) were
collected at room temperature, using a Bio-Rad FTS-60A spectrometer in
rapid-scanning mode, a liquid-nitrogen-cooled MCT detector, a
resolution of 2 cm1, triangular apodization, and one
level of zero filling. Base-line single-beam spectra were recorded
immediately before the addition of 10 µg of lipoprotein complex or
lipid-free apoprotein to a continuously stirred buffer subphase (30 mM HEPES in D2O, pD 7.4). The volume of the
subphase was 6.0 ml, making the protein concentration approximately 60 and 70 nM for apohA-1 and apo
(1-43)A-1, respectively. The supported monolayers consisted of 80 mol % DMPC and 20 mol % DMPS. Phospholipid concentrations were assayed colorimetrically (31).
Sets of parallel and perpendicular spectra were analyzed simultaneously
using "IRfit" (24).
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RESULTS |
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When hA-1 and (1-43)A-1 lipoprotein complexes were injected
into a subphase buffer containing 1 mM Ca2+,
spectroscopic signals developed and slowly reached an intensity plateau
over approximately 60 min. Because the total cross-sectional area of
all the complexes (assuming 130 nm diameter) corresponding to 10 µg
of protein is less than the surface area of the trough (approximately
90 cm2), the signal plateau most likely corresponds to
adsorption of the entire injected sample onto the monolayer, and it is
unlikely the lipoprotein complexes have "stacked" on the support
membrane. Irrespective of whether the complexes are stacked, however,
conclusions drawn below with respect to the relative orientation of
protein and lipid components remain valid.
Representative infrared spectra of the hA-1 lipoprotein complex are
shown in Fig. 2, a and
b; spectra of the
(1-43)A-1 complex are similar. The lipid components are seen as
bands at 2920 cm
1 from the antisymmetric CH2
stretch, at 2850 cm
1 from the symmetric CH2
stretch, and at 1740 cm
1 from the ester C = O
stretch. Amide I from the protein component is maximal at 1645 cm
1, consistent with an
-helical conformation, while
amide II at 1550 cm
1 (off scale in Fig. 2b) is
not detectable. The absence of an amide II band at 1550 cm
1 indicates that H
D exchange is virtually
complete.
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When lipid-free apohA-I or apo(1-43)A-1 was introduced into a
subphase buffer containing 1 mM Ca2+, an amide
I signal developed over a similar period of time as did the lipoprotein
complexes, and their amplitudes were comparable with those of the
complexes. The amide I absorption maximum occurred at 1645 cm
1, but there were no characteristic lipid absorption
signals (Fig. 2c). Bands originating from the supported
monolayer are not seen because they are part of the background spectrum
collected prior to introduction of the complexes.
When calcium was omitted from the subphase, the signal arising after the introduction of lipoprotein complexes into the subphase was negligible. This demonstrates that freely diffusing complexes do not contribute to the spectroscopic signal. In contrast, omitting Ca2+ from the subphase buffer reduced the signal from the apoproteins by only 50%. This is important control information, because it indicates that the preparation of lipoprotein complexes used in these experiments is effectively free of apoprotein. If this were not true, we would have detected an amide I absorption when the lipoprotein complexes were examined in the absence of Ca2+.
From the polarized absorption spectra, dichroic ratios
Rz = A
/
A
were
evaluated using integrated areas of characteristic absorption bands,
A, as described elsewhere (24). Dichroic ratios were
converted to order parameters, S(Rz), according to Equations 1-3.
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(Eq. 1) |
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(Eq. 2) |
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(Eq. 3) |
S(Rz) values between 0.45 and
0.41
were obtained for the symmetric methylene stretching mode at ~2850
cm
1 in the lipoprotein complexes (Table
I). S
=
0.5,
because this mode is oriented 90° from the acyl chain axis, and
S
< 1.0, because there will
always be some mosaic spread due to imperfections in the crystal
support. Therefore the molecular order of the lipid acyl chains in the
complexes S
= S(Rz)/S
·S
)
0.82. This is close to the maximum possible value of 1.0, and it
indicates that the discoidal lipoprotein complexes are highly ordered
on the monolayer surface with their acyl chains oriented perpendicular
to the surface. In the same spectra, we find that the antisymmetric
methylene stretching mode (at 2920 cm
1) is also highly
ordered and similarly oriented.
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S(Rz) was 0.19 for the amide I band at
~1650 cm-1 in the heterogenous apohA-I complexes,
0.22
for the apo
(1-43)A-I complexes, and
0.28 for the homogeneously
sized small complexes (Table I). The difference between the
heterogenous apohA-I complexes and the homogeneously sized small
complexes is statistically significant, and it suggests that the first
43 residues of apohA-I may not be aligned with the remainder of the
protein. The difference between the heterogeneous and the homogeneous
apo
(1-43)A-I complexes is not statistically significant, and
differences in the order of the methylene stretching modes suggests
that the appearance of some change in amide I transition order may be
due to the smaller complexes simply being more uniformly oriented.
Estimates of S
range from 0.53 to 0.83 for
right-handed
-helices (30), and S
> 0.82 from the preceding paragraph. Therefore
0.5
S
0.28 for
-helices in the heterogenous
complexes, and
0.5
S
0.34 for the
smaller homogeneous complexes.
When molecular orientations are uniform, order parameters may be
re-interpreted as "tilt angles" (30). This view assigns a tilt
angle between 0° and 20° from the membrane normal to the lipid acyl
chains and between 67° and 90° to the protein helices.
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DISCUSSION |
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These results have several important implications. First, they
show that lipid molecules within these complexes are highly ordered, as
in a bilayer membrane with its surface oriented parallel to the
monolayer support. Second, they show that the protein component of
these complexes is preferentially oriented parallel to the surface of
the lipid bilayer as in the belt model (Fig. 1, left). Third, they show that complexes made with apo(1-43)A-I (for which a
crystal structure is available) yield results that are similar to those
made with apohA-I. The apo
(1-43)A-I crystal structure is a
homotetramer of predominantly helical amphiphilic segments in the shape
of a bent elliptical ring. With simple and plausible manipulations, one
may transform a pair of these segments into a circular ring (10) with a
hydrophobic inner circumference and a diameter of 7.8 nm (the same as
that of the small homogeneously sized complexes examined in this
report). A ring of this size is well suited to encircle 60 lipid
molecules per protein segment in the manner prescribed by the belt
model and to explain all of the experimental results reported herein.
Our measurements were made on complexes under conditions that do not denature the protein or disrupt the complexes (25). In contrast, the earlier study of apolipoprotein A-I on which the picket fence model has been based was performed on complexes that had been dried onto a crystal surface as a multibilayer film (13). By drying the complexes, one forfeits control over conditions such as ionic strength and pH and risks denaturing the protein and/or disrupting the complexes, because the hydrophobic effect is largely responsible for their stability. Although the film is partially rehydrated by exposure to water vapor, relatively few water molecules are actually present (32), and preparations of this type yield spectroscopic results that differ from lipid monolayers and bilayers in ways that suggest denaturation and/or aggregation (22).
In summary, our results unambiguously support the belt model of
lipoprotein A-I structure and refute the picket fence model (Fig. 1).
The data were obtained using an approach that combines internal
reflection infrared spectroscopy and Langmuir film balance technology
and is advantageous because it provides an abundance of quantitative
structural information about protein-lipid interactions under
conditions that are unlikely to disrupt the complexes or denature the
protein component.
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ACKNOWLEDGEMENTS |
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We acknowledge the technical assistance of Ken Judge, Wendy Yang, Henrietta Turner, and Dorlan Kimbrough.
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FOOTNOTES |
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* This research was supported by grants from the NIH-NIGMS and the American Heart Association (to P. H. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Dept. of Pharmacology, University of Pennsylvania, 3620 Hamilton Walk, Philadelphia, PA 19104-6084. Tel.: 215-898-9238; Fax: 215-573-2236; E-mail: axe{at}pharm.med.upenn.edu.
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ABBREVIATIONS |
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The abbreviations used are: apohA-I, Human apolipoprotein A-I; DMPC, dimyristoylphosphatidylcholine; DMPS, dimyristoylphosphatidylserine; PBS, phosphate-buffered saline; PATIR-FTIR, polarized attenuated total internal reflection Fourier-transform.
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REFERENCES |
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