Proc. Nat. Acad. Sci. USA Vol. 72, No. 6, pp. 2270-2273, June 1975

Neutron Scattering Study of Human Serum Low Density Lipoprotein (solvent exchange)

H. B. STUHRMANN*, A. TARDIEUt, L. MATEUt, C. SARDETt, V. LUZZATIt, L. AGGERBECK4, AND A. M. SCANJUt * Institut far physikalische Chemie der Universitat Mainz, 6500 Mainz, Germany; tCentre de Gen6tique Moleculaire, C.N.R.S., 91190 Gifsur-Yvette, France; and $ Departments of Medicine and Biochemistry, The University of Chicago, Pritzker School of Medicine, Chicago, Ill. 60637

Communicated by Max F. Perutz, March 20, 1975 ABSTRACT Regions of different proton density in the low density lipoprotein (LDL) particle from human serum have been determined by neutron scattering. From measurements in various H20/D20 mixtures, the LDL particle appears to be quasi-spherical, with the centers of gravity of the hydrocarbon and polar regions coinciding. The average volume occupied by a particle was found to be 3.2 X 106 Al, with the volume fraction occupied by the hydrocarbons being 60%. The radius of gyration of the hydrocarbon region was 64 A, while that of the polar region was 100 A. Consequently, the core of LDL is predominantly occupied by the hydrocarbon chains, while the outer shell is sparsely occupied by protein emerging from the lipid core.

-0.372 X 10-11 cm. Its heavier isotope, 'H or D, is characterized by b = +0.66 X 10-12 cm. The scattering lengths of carbon and oxygen are rather close to that of (leuterium, while nitrogen exhibits a somewhat larger b. Thus, mixtures of H20/D20 cover a much wider range of scattering densities than do salt or sugar solutions in x-ray work (14). The mean scattering density of a mixture of 8% D20 in H20 vanishes. In such a medium neutron scattering of the dissolvedl macromolecules "in vacuo" can be observed. This paper presents the results of an attempt to gather precise information regarding the arrangement of the lipid and protein components within LDL by neutron scattering techniques and will also elucidate sonic typical features of

Human serum lipoproteins are particles in which serum lipids are associated with characteristic polypeptides in discrete species generally defined by their hydrated density (1). In addition to playing a major role in lipid transport and energy metabolism (2), lipoproteins may be involved in the maintenance of cell membranes (3) and in the regulation of lipid biosynthesis (1, 4). The study of lipoproteins is of particular importance because of several diseased states associated with alterations in their concentration and/or structure (5). Low density lipoproteins (LDL) are the predominant species in the density range 1.019-1.063 g/ml in human serum (1-4). These particles, which appear to be roughly spherical in shape (6, 7) and to have an average molecular weight of 2 to 3 X 106 (8), are thought to be complex associations of 21% protein, 22% phospholipids, 8% free cholesterol, 37% cholesterol esters, and 11% triglycerides (1-4). How these lipid and protein components are arranged in LDL remains elusive, although several models have been suggested (6, 7, 9-11). Small angle x-ray scattering of solutions of varying electron density may provide definitive information about LDL structure (12); however, a precise structural analysis requires great accuracy in experimental data collected over an extended range of solvent densities. This has been accomplished recently using the novel, position-sensitive x-ray detector (manuscript in preparation). An alternative approach, to a large extent complementary to small angle x-ray scattering, is offered by neutron scattering (13). Unlike x-rays, neutrons are scattered by nuclei. The scattering lengths, b, of the nuclei are relatively small and comparable to the atom form factors of the "light" atoms in x-ray scattering. However, some nuclei have negative scattering lengths. The most prominent example is 'H: b =

neutron scattering by solutions (15). EXPERIMENTAL PROCEDURE

Human serum LDL (density 1.019-1.063 g/ml) was isolated by preparative ultracentrifugation from blood obtained from healthy, fasting (18 hr), Caucasian male donors, 20-30 years of age, group A, Rh-positive (16). Following isolation, LDL was extensively dlialyzedl in the dark at 40 against 150 mMI NaCl, 1 mM\ EDTA, pH 7.4. Subsequently a portion of the LDL was (lialyzed against D,0 and then 11 mixtures were made by combining the solutions of LDL in D20 and LDL in H20. In addition, a series of solutions containing decreasing concentrations of LDL were prepared in H20/D20 mixtures containing 97%, 59%, and 0% D20. All scattering curves were extrapolated to infinite dilution of LDL. The neutron scattering experiments were (lone at the Institute iMax von Laue-Paul Langevin at Grenoble. Neutrons from the 57 AMW high flux reactor were moderated by liquid deuterium, monochromatized by a helical slot selector, scattered by a sample volume of 0.5 ml, and detected by a two-dimensional array of 64 times 64 counters arranged on a square of 64 cm height. The wavelength spectrum was centered at 3.7 A and had a total half width of 1.7 A. The correction of the neutron scattering curves for the influence of this broad wavelength distribution turned out to be inadequate. It resolved quite clearlv the maxima of the curves (Fig. 1); however, the depth of the minima often remained quite inaccurate. In order to cover a sufficiently broad range of scattering angles we placed the detector at 0.66, 2, and 10 m from the sample. The neutron flux passing the sample area of 2 cm2 varied from 106 to 108 neutrons/'s depending on the angular resolution.

Abbreviation: LDL, low density lipoprotein.

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Neutron Scattering of Low Density Lipoprotein

(1975)

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-41

.B

.02 .0% .06 .08 .10 .12 .14 .16 -ae [.4?1] FIG. 1. Neutron scattering of LDL in various H20/D20 mixtures: *, 59% D20; 0, 45% D20; O, 30% D20; 0, 15% D20; 0, 0% D20. The intensities are given on absolute scale and correspond to differential cross sections of LDL. The curves are corrected for polychromatism and collimation distortions. RESULTS AND DISCUSSION

Neutron scattering experiments were performed in the presence of variable amounts of D20 (Fig. 1). When the scattering density of the solvent is diminished, the excess scattering density of the LDL particle with respect to the solvent will increase in those regions which are not, or only partially, accessible to the solvent (17). The local increase of the contrast is described by pc(r). This function, pc(i), defines the shape of the particle if no part of the particle can be penetrated by the solvent (18, 19). In this case pc(r) would have the value 1 inside the particle and it would vanish outside the boundaries of the dissolved particle. In general, a certain degree of inner solvation is known to occur; formally, this solvation can be taken into account by allowing pc(r) to be smaller than one. H/D exchange of dissociating protons influences pc(r) in the same way. That part of the excess scattering density of the solute which does not depend on the scattering density of the solvent is called ps(r). ps(r) corresponds to the fluctuations of scattering density within the solute particle about its mean and its integral over the particle is zero. The overall contrast p is given by P = PLDL - Psolvent

[1]

where p denotes a mean scattering density. ps(r) can be measured directly at vanishing contrast p = 0. The excess scattering density, p(r), with respect to the solvent is then

p(r)

=

ppc(r) + Ps(r)

[2]

A (K)

PAc(i)

+ A s(v)

[3]

where K is the momentum transfer of the scattered neutron, K = (47r/X) -sin 0, 20 = scattering angle. X is the wavelength of the incident and scattered neutrons.

-2

-4 i

2

0

[lo- cmiI

--be

FIG. 2. The radius of gyration R of LDL is strongly influenced by the contrast p. AR2 has no dimension.

For a quasi-spherical particle like LDL, the spherically averaged part of the structure, p (r), is predominant at small K

(17, 18): = (A (,))2 = (P(AC(oc)) + (AS(K))) 2 [4] The square root of the extrapolated zero-angle scattering of LDL turns out to be a linear function of the volume fraction of D20 of the solvent. Therefore, a significant heterogeneity of scattering density among LDL particles can be excluded (14). Zero-angle scattering vanishes in a solvent containing 15% D20; therefore, the average density of LDL (PLDL in Eq. 1) is 0.48 X 101° cm-2. The scattering density of LDL is considerably lower than that of proteins (2.0 X 1010 cm-2) because of the presence of the hydrocarbon chains (Phydr = -0.3 X 10° cm-2). The polar groups of the lipids have scattering densities which are only slightly lower than that of the proteins. According to the chemical composition of LDL, we assume that the "polar" regions (polar groups of the lipids and proteins) have a mean scattering density Ppol = 1.7 X. 1010 cm-2. The volume fraction x of the hydrocarbon regions can be estimated:

I1(K)

-0.3x + 1.7(1 - x) = 0.48. About 60% of the LDL particle appears to be occupied by the hydrocarbon chains. This value is 8% higher than that estimated from the chemical data (13). A quantitative idea of the arrangement of the hydrocarbon and the polar components can be deduced from the dependence of the radius of gyration R on the contrast p. It has been shown (12, 14) that, within the validity of Eq. 2:

pR2

and it gives rise to the amplitude =

-6

=

PRC2 +

a +

(/p.

[5]

The experimental points plotted in Fig. 2 clearly show that pR2 is linearly dependent on p and that (3 = 0. Several conclusions can be drawn and various parameters be determined from this observation. (a) The fact that (3 = 0 indicates that the position of the center of gravity of p(r) is independent of the density of the

Biophysics: Stuhrmann et al.

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Proc. Nat. Acad. Sci. USA 72

80 r [A]

ae[KI FIG. 3. The spherically averaged amplitudes of LDL. -, A AC(K) with A = l09 cmM2; broken line, AS (K). The ordinate is

given in 10-10 cm. Ac(O)

=

2.8 X 10-9 cm.

solvent. In other words, the centers of gravity of the polar and hydrocarbon regions coincide. (It is worth noting that in a particle formed by two regions of different density, the parameter # defines the distance between the centers of gravity of the two regions.) (b) The slope of the straight line defines the value of the radius of gyration of the solvent-excluding solid, pc(r): Rc= 80X. (c) The intercept at p = 0 defines the value of a, and thus the second moment of the internal fluctuations of the scattering density ps(r): a = fps(r)r2d&r/fpc(r)dar.

[6]

Since a = 2.8 X 10-i and fpc(r)d~r = 3.2 X 106 A' (see below), fps(r)r2dar = 9.0 X 108 A'. Such a large value, about two orders of magnitude larger than in most proteins, indicates the presence of conspicuous long range fluctuations of the scattering density inside the LDL particles. (d) The values of PR2, interpolated at the densities corresponding to the polar and to the hydrocarbon regions (thus the conditions in which one or the other region is erased), allow one to determine the radius of gyration of the hydrocarbon and of the polar regions, respectively (see Eq. 5):

Rpol

=

(Rc' + a/(pLDL

-

Phydr))/2

100 A

Rhydr = (RC2 + a/(PLDL Ppol))l/2 = 64 A The difference between the two radii of gyration shows that the hydrocarbon chains are located preferentially in the inner part and the proteins in the outer shell of the LDL -

particles. A further step in the analysis is based upon the observation that, to a 50 A resolution, the LDL particle appears to exhibit a spherical symmetry. This is shown by the linear dependence of the square root of I(K) on the scattering density of the solvent (see Eq. 4). In this case V/I(K) can be decomposed into the sum of Ac(K) and AS(K) (see Fig. 3), and the spher-

(197-6)

120

-'P

FIG. 4. The radial scattering density functions of LDL. 0, f5pc(r) with fi = 1010 cm-2; *, ps(r); solid line, pv(r). pc(r) gives the radial distribution corresponding to the shape of the particle considered as an object with uniform average density. ps(r) gives the radial distribution of the fluctuations of scattering density within the particle about the mean density. pv(r) gives the radial scattering distribution of the particle in vacuo.

ically symmetric scattering density functions pc(r) and ps(r) can be determined by Fourier transformation of Ac(K) and AS(K). The results are shown in Fig. 4. ps(r), the radial distribution of the internal fluctuations of scattering density with respect to its average, PLDL, displays a low density core due to the high concentration of hydrocarbon chains and a high density outer shell due to the proteins. pc(r), the radial distribution of the shape function, is almost constant up to r = 80 A, and then it decreases monotonously and vanishes at r = 125 A. Several reasons can explain the departure of pc(r) from a step function that would correspond to a spherical volume. One is the influence of H/D exchange and of the presence of solvent molecules inside the LDL particles. Another reason is the effect of truncation on the Fourier transform of Ac(K). And a third one may be the presence of a rugged surface. This last effect is confirmed by the fact that the radial scattering distribution in vacuo, pv(r), reaches a maximum of 0.6 X 1010 cm-2 (see Fig. 4), whereas the expected scattering density of a protein is 2.0 X 1010 cm-2. It may thus be inferred that only one-third of the outer layer is occupied by proteins emerging from the lipid core. LDL has also been the subject of kinetic studies of H/D exchange. One part of LDL in H20 was diluted by four parts of D20. During the observation period, from 1 min to half an hour after mixing, zero-angle scattering decreased by 3%. Only one half-time of about half an hour could be obtained from the kinetic measurements. A comparison with corresponding experiments on other proteins might be useful; in kinetic studies of H/D exchange of myoglobin a decrease of small angle scattering of about 20% has been observed under the same experimental conditions (14). About 20% of dissociating protons are exchanged during this period (20). In the case of LDL the contrast in a final H20/D20 mixture

Proc. Nat. Acad. Sci. USA 72

(197-6)

containing 80% D20 is two times higher than with proteins in the same solvent and the concentration of polar groups is lowered by the presence of 60% hydrocarbon. From these data a decrease of 4% of the zero-angle scattering of LDL is expected, which agrees reasonably well with our measurements. If only 20% of the dissociating protons are supposed to exchange during the measuring period, the total change in contrast would be 7%. The mean value of pc(r) would be 0.93 due to H/D exchange. pc(r) will be close to 1 in the central part and considerably lower values will be found in the outer hydrophilic regions of the LDL molecule. Correcting pc(r) for the influence of H/D exchange, the volume, V, is obtained by integration over pc(r). The resulting Vc = 3.2 X 106 AA must be regarded as a minimum value because a possible influence of solvation on pc(r) cannot be distinguished by our method. From Vc and the partial specific volume, v = 0.971 cm3 g'-, (6, 21) the molecular weight, M = (0.971) X (6.02 X 1023) X (3.2 X 10-18) = 1.9 X 106, is obtained. A somewhat higher molecular weight for LDL, M = 2.2 X 106, is reported by Scanu and Wisdom (1). The kinetic studies do not indicate a change of the radius of gyration exceeding the experimental error of less than 1%. CONCLUSIONS

The main results of our neutron scattering studies can be summarized as follows: Average scattering density of LDL: PLDL = 0.48 X 1010 cm-2. Volume fraction occupied by hydrocarbons: 60%. The centers of gravity of the hydrocarbon and the polar region (protein plus polar groups of the lipids) coincide. Volume occupied by one particle: Vc = 3.2 X 106 Xg. Radius of gyration of the shape function: Rc = 80 K. Radius of gyration of the hydrocarbon region: Rhydr = 64 K. Radius of gyration of the polar region: Rp.1 = 100 K. Second moment of the scattering density fluctuations: f ps(r)r2d3r = 9.0 X 103.3 The LDL particles are quasi-spherical. The core is predominantly occupied by the hydrocarbon chains. The outer shell is sparsely occupied by protein molecules emerging from the lipid core. It is rewarding to note the wealth of information which can be obtained by small angle neutron scattering experiments when full advantage is taken of H/D replacement. A more

Neutron Scattering of Low Density Lipoprotein

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refined interpretation will be possible when recent x-ray scattering results are taken into account. We are grateful to Dr. K. Ibel for kindly assisting in the neutron scattering experiments and to the former director of the Institut Max von Laue-Paul Langevin, Dr. B. Jacrot, whose enthusiasm encouraged us throughout. This work was supported by the Institut Laue-Langevin (H.B.S., Institut Max von Laue-Paul Langevin Research Participant from September 1971 to February 1973); D116gation Gen6rale A la Recherche Scientifique et Technique (A.T., L.M., C.S., and V.L.); U.S. Public Health Service Grants H.D. 00001 (L.A.), and (HE-08727); the American Heart Association (no. 70-7.53); the Chicago and Illinois Heart Associations (C71-6); and the U.S. Atomic Energy Commission [Contract AT(11-1)-69] (A.M.S.). A.M.S. is recipient of a Research Career Development Award (no. HE-24,867) from the U.S. Public Health Service. 1. Scanu, A. M. & Wisdom, C. (1972) Annu. Rev. Biochem. 41, 703-730. 2. Scanu, A. M. (1965) Adv. Lipid Res. 3, 63-138. 3. Schumaker, V. N. & Adams, G. H. (1969) Annu. Rev. Biochem. 38, 113-136. 4. Scanu, A. M. & Ritter, M. C. (1973) Adv. Clin. Chem. 16, 111-151. 5. Fredrickson, D. S., Levy, R. I. & Lees, R. S. (1967) N. Engl. J. Med. 276, 32-44, 94-103, 148-156, 215-226, 273281. 6. Pollard, H., Scanu, A. M. & Taylor, E. W. (1969) Proc. Nat. Acad. Sci. USA 64, 304-310. 7. Forte, T. & Nichols, A. V. (1972) Adv. Lipid Res. 10, 1-41. 8. Scanu, A. M., Vitello, L. & Deganello, S. (1974) CRC Crit. Rev. Biochem. 2, 175-196. 9. Gotto, A. M. (1969) Proc. Nat. Acad. Sci. USA 64, 11191127. 10. Oncley, J. L., Gurd, F. R. N. & Melin, M. (1950) J. Am. Chem. Soc. 72, 458-462. 11. Cook, W. H. & Martin, W. G. (1962) Can. J. Biochem. Physiol. 40, 1273-1285. 12. Mateu, L., Tardieu, A., Luzzati, V., Aggerbeck, L. & Scanu, A. M. (1972) J. Mol. Biol. 70, 105-116. 13. Schelten, J., Schlecht, P., Schmatz, W. & Mayer, A. (1972) J. Biol. Chem. 247, 5436-5441. 14. Stuhrmann, H. B. (1974) J. Appl. Crystallogr. 7, 173-178. 15. Schmatz, W., Springer, T., Schelten, J. & Ibel, K. (1974) J. Appl. Crystallogr. 7, 96-116. 16. Scanu, A. M., Pollard, H. & Reader, W. (1968) J. Lipid Res. 9, 342-349. 17. Harrison, S. C. (1969) J. Mol. Biol. 42, 457-483. 18. Stuhrmann, H. B. (1973) J. Mol. Biol. 77, 363-369. 19. Stuhrmann, H. B. (1970) Z. Phys. Chem. 72, 185-198. 20. Englander, S. W. & Staley, R. (1969) J. Mol. Biol. 45, 277295. 21. Pollard, H. & Devi, S. K. (1971) Biochem. Biophys. Res. Commun. 44, 593-599.

Neutron scattering study of human serum low density lipoprotein.

Proc. Nat. Acad. Sci. USA Vol. 72, No. 6, pp. 2270-2273, June 1975 Neutron Scattering Study of Human Serum Low Density Lipoprotein (solvent exchange)...
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