From the
The secondary structure of human apolipoprotein B at 37 °C
is estimated to be 24%
Human serum low density lipoprotein (LDL)
ApoB is one of the largest proteins known, containing 4536 amino
acid residues, and is extremely insoluble in aqueous media. This has
hindered the understanding of the structural basis of apoB function,
although its primary structure has been known for some time
(1, 2) . Infrared spectroscopy (IR) was first applied to
the study of apoB by Scanu et al. (7) to examine its
thermal behavior. Later, IR was applied to apoB studies qualitatively
using resolution-enhanced techniques
(8) or quantitatively
using curve fitting of deconvolved spectra
(9) . Also, the
structure of the lipid-attached protein has been described by IR in
combination with proteolytic digestion
(10) .
The effect of
temperature on protein conformation has been extensively studied for a
variety of proteins using IR
(11, 12, 13, 14, 15, 16) .
However, when studying the thermal dependence of LDL structure, more
attention has been paid to the lipid
(3, 4, 5) than to the protein moiety
(17, 18) . We have
investigated the structure of apoB using a method that provides
accurate conformational values
(16) . The lipoprotein
environment has been modified through changes in temperature and ionic
strength without modification of the particle composition, and the
resulting variation of protein structure has been characterized.
To study
the effect of temperature, samples in D
The assignment of these bands has been carried out
previously
(16, 21) . The band at 1656 cm
From
the combined results in H
At 30 °C, a sharp shift of 2 cm
The thermal event at 75 °C is associated with irreversible
protein denaturation
(3, 4) . Its most characteristic
feature is the disappearance of the band at 1693 cm
The amide I band component
at 1618 cm
ApoB secondary structure is not yet well established, and
different structures have been proposed, even using the same technique.
Thus, from CD spectra, the following values have been given;
Information on the apoB attachment to the lipid surface can be
obtained by studying the unique characteristics of some LDL protein IR
bands. The most striking feature of the amide I band is the component
at 1618 cm
After irreversible thermal denaturation,
the particles appear to be disrupted concomitant with a release of
cholesteryl esters and protein aggregation
(3) . This is
consistent with the disappearance of the band at 1693 cm
In conclusion, this study provides new data on the structure of apoB
in LDL from human plasma and shows how factors not altering particle
composition can lead to marked variations in protein conformation. In
the range of physiological temperature, apoB conformation appears to be
rather resistant to environmental changes. The present approach can be
applied to elucidate how factors affecting core composition, such as
diet, influence protein conformation and to determine the mechanism by
which factors known to influence the LDL-receptor interaction, such as
oxidation, exert their action.
The
figures have been rounded off to the nearest integer.
We thank the Universidad del Pas Vasco for a visiting
professorship (to G. P.).
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-helix, 23%
-sheet, 6%
-turns,
24% unordered structure, and 24% ``
-strands,''
characterized by a band around 1618 cm
, and
consistent with extended string-like chains in contact with the lipid
moiety not forming
-sheets. When cooled to a temperature below the
cholesteryl ester transition at 30 °C, the ordering of the low
density lipoprotein core results in reversible changes in the protein
conformation, decreasing the apparent amount of
-helix,
-strand, and unordered structure below 30 °C and increasing
-sheet and
-turns. Lowering the ionic strength affects the
core-associated transitions, shifting their temperature from 30 to 20
°C, and modifying protein conformation below the transition. An
additional thermal event is observed at 75 °C, leading to
irreversible protein denaturation. In the broad temperature range
between the 30 and 75 °C transitions, apolipoprotein B is stable
toward both temperature and ionic strength changes. After thermal
denaturation, the protein retains a certain degree of ordered
structure.
(
)
is a major carrier of serum cholesterol in humans. It is
described as a spherical particle containing a hydrophobic core of
cholesteryl esters and triglycerides surrounded by an amphipathic
monolayer of phospholipid and cholesterol in which a single molecule of
apoB is located
(1, 2, 3) . The core-associated
lipids have been shown to undergo an order
disorder transition
near human body temperature, the actual transition temperature being
determined by the lipid composition
(4, 5, 6) .
Preparation of Lipoproteins
Blood (300
ml) of fasting females or males was poured over 15 ml of concentrated
buffer containing EDTA (1
M Tris-HCl, pH 7.4, 1 g/liter EDTA,
2 g/liter chloramphenicol) in the blood bank. Plasma was immediately
separated at 4 °C by repetitive low speed centrifugation. LDL was
obtained by sequential floating ultracentrifugation in a Kontron
Centricon T2080 ultracentrifuge using a TFT 50.38 rotor at 48,000 rpm
for 20 h at 7 °C according to the modified method of Jürgens
et al. (19) . Potassium bromide was used to adjust the
serum densities to yield the LDL fraction. LDL was isolated at d = 1.019 - 1.050 g/ml by two repetitive
centrifugations. The concentration of EDTA was kept constant (1
g/liter) through all preparative steps to prevent lipoprotein
peroxidation. Lipoproteins were dialyzed overnight in the dark against
0.1
M Tris-HCl, pH 7.4, containing 1 g/liter EDTA, then
concentrated by pressure dialysis or Amicon MDS-1 system and
subsequently filtered through a 0.45-µm filter. Isolated
lipoproteins were stored overlaid with Nat 4 °C in the
dark. Prior to IR measurements, samples were dialyzed against the
appropriate H
O or D
O buffers (0.1 or 0.01
M Tris-HCl, pH or pD 7.4 (pD = pH meter reading +
0.4) containing 1 g/liter EDTA. The purity of LDL samples was checked
by gel-agarose electrophoresis. The protein content of LDL was
determined according to Lowry with bovine serum albumin as a standard.
Infrared Studies
IR samples were measured
in a Nicolet 520 spectrometer. Sample treatment, spectra acquisition,
and resolution enhancement were performed as previously described
(11, 20) . Protein concentration was 25 mg/ml.
Quantitative information on protein structure was obtained through
decomposition of the amide I band into its constituents
(16) ,
leaving the gaussian fraction free in the first step of the fitting
procedure. Even if the values of band position and percentage area were
not affected, the fitting was improved with this procedure.
O buffer were heated
in steps of around 4 °C in the interval of 15-85 °C.
After every heating step, the sample was left to stabilize for 5 min,
and the corresponding spectrum was recorded and treated as described
above.
Apolipoprotein Structure at 37
°C
Band decomposition of the amide I band of human
plasma LDL apoB in HO and D
O media at
physiological temperature is shown in Fig. 1. The corresponding
parameters, i.e. band position, percentage area, and bandwidth
of each spectral component are displayed in Table I. The D
O
spectrum exhibits seven bands assignable to protein substructures
located at 1694, 1678, 1670, 1656, 1643, 1630, and 1618
cm
. In the H
O spectrum, six bands
located at 1695, 1683, 1670, 1653, 1632, and 1618 cm
are seen.
in D
O is undoubtedly assigned to
-helix, and the
one around 1630 cm
is assigned to
-sheet. The
bands at
1670 and 1680 cm
arise from
-turns. The band at 1643 cm
in D
O
corresponds to unordered structure; in H
O, this band is
shifted to around 1657 cm
and overlaps with the
-helix structure
(22) . It is not usual to find a protein
band at frequencies as low as 1618 cm
. In fact, in
non-denatured proteins in H
O solution, it has only been
found in apolipoproteins and assigned to ``low frequency
-sheets'''
(9) or to a
-structure less
accessible to the external solution
(8) . According to its
position, this band must be assigned to an extended structure, even
more so considering the presence of a band at 1694 cm
that corresponds to the ( 0,
) vibrational mode of
the extended chain. However, it cannot be assigned to anti-parallel
-sheet because the high frequency component does not undergo the
isotopic shift typically produced in that kind of structure by changing
the medium from H
O to D
O
(22) . A more
generalized assignment would be to
-strands, i.e. extended structures not forming
-sheet
(23) .
O and D
O
(16) ,
the secondary structure of apoB appears to be 24%
-helix, 23%
-sheet, 6%
-turns, 24% unordered structure, and 24%
-strands.
Influence of Temperature on Lipoprotein
Structure
The influence of temperature on LDL conformation
has been studied by observing the IR spectral changes when the protein
is submitted to stepwise heating in DO medium
(16) .
Fig. 2 shows the amide I band decomposition of LDL in 0.1
M pD
7.4 buffer at 17 °C ( A) and 80 °C ( B).
Opposite to what occurs in most membrane proteins, no residual amide II
is found near 1545 cm
(not shown), indicating a
complete deuterium exchange in the amide bonds of the protein.
Additional data are obtained from the amide I band components; a
temperature profile corresponding to the
-helix component (Fig. 3)
is shown as an example. In 0.1
M Tris-DCl buffer ( filled circles), two different thermal events are observed in
the range 15-85 °C, one at 30 °C and the second around 75
°C. The thermal effect at 30 °C is reversible, whereas the one
observed at 75 °C is not. The temperature behavior of band position
and percentage area for the various band components is summarized in
Table II.
or
larger is observed in the bands near 1655, 1668, and 1684
cm
, and a somewhat smaller one in the band at 1644
cm
is observed, while the maxima of bands at 1630
and 1615 cm
vary smoothly with temperature.
Important variations in the proportions of the various conformational
components also occur at 30 °C. The band corresponding to
-helix increases from 17 to 24%; the band at 1668 cm
varies from 10 to 3%, and the
-sheet band drops from 29 to
23%.
.
Parameters associated with each of the band components appear to vary
in a characteristic way (). Pronounced changes occur,
e.g. a significant decrease in the unordered conformation
(from 24 to 6%) and an increase in the band corresponding to
-sheet structure (from 21 to 31%).
shows a peculiar thermal behavior in that
it parallels the change in position of the lipid methylene stretching
vibrations at
2854 cm
(Fig. 4). This is the only
component of apoB amide I that follows this pattern.
Studies in Low Ionic Strength Buffer
The
thermotropic behavior of LDL in a low ionic strength buffer (0.01
M Tris-DCl) has also been characterized (Table III).
Fig. 3
shows the thermal profile of -helix in the low ionic
strength buffer ( open circles). The most remarkable
difference between the high and low ionic strength samples is a
decrease in the temperature of the reversible thermal event from 30 to
20 °C. The sign and extent of the shifts in band position are
rather similar at low and high ionic strength (I). Table
IV summarizes the data on the fractional distribution of secondary
structures in LDL at high and low ionic strength. The change in salt
concentration clearly affects the structure of apoB below 20 °C and
above 75 °C; however, between the transitions, the change in ionic
strength does not lead to significant conformational differences. Below
20 °C, the protein shows a higher proportion of regular
conformations, i.e.
-helix and
-sheet, under
conditions of low ionic strength. Above the thermal denaturation
temperature, however, spectra taken under low ionic strength conditions
show a lower proportion of
-helix and a higher fraction of
unordered structure than their counterparts in 0.1
M buffer
(). Thermal denaturation temperature is not greatly
affected by ionic strength. Note that a residual structure, different
at high and low ionic strength, is present after thermal denaturation,
as reported for other proteins
(12, 15, 16) .
Figure 3:
Thermal profiles of the amide I component
corresponding to -helix. Band position, percent band area, and
bandwidth are plotted as a function of temperature for preparations of
apoB in D
O medium. Filled circles correspond to spectra taken in 0.1
M Tris-DCl, pD 7.4,
and open circles correspond to spectra taken in 0.01
M Tris-DCl, pD 7.4.
40%
-helix,
20%
-sheet, and
40% random coil
(24) or 25%
-helix, 40%
-structure, and 35% disordered
regions
(25) . A content of 43%
-helix, 21%
-sheet
structure, 20% random coil structure, and 16%
-turns has been
postulated using a secondary structure prediction algorithm
(26) . A quantitative IR study found 21%
-helix, 41%
-sheet, 19%
-turns, and 19% random coil
(9) . The
latter results and some of the CD data are close to those presented in
this paper, taking into account that the band at 1618 cm
was assigned previously to ``low-frequency
-sheet
structure.'' Another factor contributing to the small
discrepancies between the IR values is that here the original and not
the deconvolved spectrum is decomposed, since in deconvolved spectra
the area ratios are fully preserved only when bandwidths are alike
(27) , which is not always the case in practice. Moreover, our
approach combining IR band fitting and temperature has proved to be
sensitive to small changes in protein conformation
(16) .
. In proteins, a band at a lower frequency
than the one associated to
-sheet structures (1630
cm
) was first described in H
O buffer for
concanavalin A
(28) ; it was then assigned to a
``
-edge'' structure, formed by extended peptide
structures located toward the end of the main strands, or on the outer
strands of the
-sheets. Also, a similar band has been reported in
cases of protein aggregation produced after thermal denaturation in
D
O buffer
(14, 29) and has also been
detected in dimeric
(16) but not in monomeric cytochrome
oxidase
(15) being attributed to intermolecular
-sheet or
protein-protein contacts. The proposed structure consists in all cases
of an extended chain not forming a
-sheet with other chains but
interacting via a different hydrogen bonding pattern with extended or
coiled structures from the same or a different polypeptide chain.
Moreover, its thermal behavior is different from that of other apoB
amide I components and follows a pattern similar to the methylene
stretching of the lipid moiety (Fig. 4). This would support an
assignment of this component as a protein structure penetrating the
monolayer and establishing hydrophobic interactions deeper than the
outer charged shell. The existence of protein regions inside the
monolayer has been deduced from controlled proteolysis that shows
non-accessible regions of the protein molecule tightly associated with
LDL
(26) . The assignment of the 1618 cm
band
to this protein fraction interacting with lipids is also sustained by
the observed increase in this component after protease treatment
(10) . The higher strength of hydrogen bonds within the
hydrophobic milieu would account for the
7 cm
difference existing between the position of this component in
lipoprotein and in concanavalin A (1625 cm
) or
cytochrome c oxidase (1624 cm
). The amount
of extended chain interacting with the lipid moiety (18-24%, see
Table IV) would involve between 820 and 1090 peptide bonds. Assuming a
displacement of 3.47 Å per residue, the length of the chain would
wrap more than four times around the equator of the LDL particle.
Therefore, these results would match a disposition of the protein as a
long flexible string as suggested by immunoelectron microscopy
(30) . However, our studies can not distinguish the putative
existence of a number of domains that would be connected by the
flexible string. A recent re-evaluation of LDL structure
(31) gives a picture of the protein as penetrating about half of
the phospholipid monolayer and presenting, perhaps, a three-domain
structure.
Figure 4:
Band position versus temperature
plot for two LDL infrared spectral features. Band positions correspond
to the lipid CHsymmetric stretching (
2854
cm
) and to the protein
-strand conformation
(
1618 cm
). Filled circles correspond to spectra taken in 0.1
M Tris-DCl, pD 7.4,
and open circles correspond to spectra in 0.01
M Tris-DCl, pD 7.4.
The reversible thermotropic protein transition at 30
°C is produced at a temperature similar to the previously described
order-disorder transition of the core-located cholesteryl esters
(3, 4) involving a core-modulated protein
conformational change. The decrease in the transition temperature
produced by a low ionic strength buffer is compatible with an increase
in the equilibrium magnetization of the protons belonging to the mobile
fraction of the particle interior at 22 °C observed by NMR
(32) . The transition involves an increase in -helix and
unordered structure at the expense of
-sheet and
-turns, and
the same effects are observed at both high and low ionic strength
samples albeit with different intensities. In general, changes in
protein conformation at both thermal transitions have opposite signs,
e.g. a decrease in the proportion of
-sheet (1630
cm
) at the low temperature transition versus an increase at 75 °C (Tables II and III). However, the region
between the transitions, containing the human body temperature, is
remarkably stable, suggesting some degree of homeostasis at the level
of LDL structure. The observed alterations in protein conformation
induced either by temperature or by ionic strength confirm the
surface-core correlation postulated by NMR and ESR
(32, 33) .
and the change in trend of the band at 1618
cm
, giving rise to a classical pattern of protein
aggregation after denaturation in a D
O buffer
(16) .
Table: IR spectrum of LDL apolipoprotein at 37
°C in a 0.1
M Tris buffer, pH or pD 7.4
Table: Temperature-induced changes in the
components of human low density lipoprotein infrared spectrum amide I
band in 0.1 M Tris buffer, pD 7.4
Table: Temperature-induced changes in the amide I
band components of human low density lipoprotein infrared spectrum in
0.01
M Tris buffer, pD 7.4
Table: Percent values of the secondary structure
components of LDL apolipoprotein measured in different ionic strength
buffers below the first thermal event, between both thermal
transitions, and above the second thermal transition
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.