©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Surface-Core Relationships in Human Low Density Lipoprotein as Studied by Infrared Spectroscopy (*)

Sonia Bauelos (1)(§), José Luis R. Arrondo (1)(¶), Félix M. Goi (1), Greta Pifat (2)

From the (1) Departamento de Bioqumica, Universidad del Pas Vasco, Apartado 644, E-48080 Bilbao, Spain and the (2) Ruder Bo&;kovi&; Institute, Bijeni&;ka 54, 41001 Zagreb, Croatia

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The secondary structure of human apolipoprotein B at 37 °C is estimated to be 24% -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.


INTRODUCTION

Human serum low density lipoprotein (LDL)() 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) .

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.


MATERIALS AND METHODS

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 HO or DO 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.

To study the effect of temperature, samples in DO 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.


RESULTS

Apolipoprotein Structure at 37 °C

Band decomposition of the amide I band of human plasma LDL apoB in HO and DO 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 DO spectrum exhibits seven bands assignable to protein substructures located at 1694, 1678, 1670, 1656, 1643, 1630, and 1618 cm. In the HO spectrum, six bands located at 1695, 1683, 1670, 1653, 1632, and 1618 cmare seen.

The assignment of these bands has been carried out previously (16, 21) . The band at 1656 cmin DO is undoubtedly assigned to -helix, and the one around 1630 cmis assigned to -sheet. The bands at 1670 and 1680 cmarise from -turns. The band at 1643 cmin DO corresponds to unordered structure; in HO, this band is shifted to around 1657 cmand 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 HO 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 cmthat 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 HO to DO (22) . A more generalized assignment would be to -strands, i.e. extended structures not forming -sheet (23) .

From the combined results in HO and DO (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.

At 30 °C, a sharp shift of 2 cmor larger is observed in the bands near 1655, 1668, and 1684 cm, and a somewhat smaller one in the band at 1644 cmis observed, while the maxima of bands at 1630 and 1615 cmvary 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 cmvaries from 10 to 3%, and the -sheet band drops from 29 to 23%.

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. 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%).

The amide I band component at 1618 cmshows 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 DO 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.




DISCUSSION

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; 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 cmwas 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) .

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. In proteins, a band at a lower frequency than the one associated to -sheet structures (1630 cm) was first described in HO 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 DO 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 cmband 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 cmdifference 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) .

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 cmand the change in trend of the band at 1618 cm, giving rise to a classical pattern of protein aggregation after denaturation in a DO buffer (16) .

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.

  
Table: IR spectrum of LDL apolipoprotein at 37 °C in a 0.1 M Tris buffer, pH or pD 7.4

The figures have been rounded off to the nearest integer.


  
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



FOOTNOTES

*
This work was supported in part by Grants 161/92 from the University of the Basque Country and PB91-0441 from Direccion General de Investigacion Cientifica y Tecnica and the Ministry of Science and Technology, Republic of Croatia. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
A recipient of a fellowship from the Basque Government.

To whom correspondence should be addressed. Tel.: 34-4-464-7700 (ext. 2407); Fax: 34-4-464-8500.

The abbreviations used are: LDL, low density lipoprotein; apoB, apolipoprotein B-100; IR, infrared spectroscopy.


ACKNOWLEDGEMENTS

We thank the Universidad del Pas Vasco for a visiting professorship (to G. P.).


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.