TIIE JOURNAL OF BIOLOGICAL CHEMISTBY Vol. 247, Eo. 8, Issue of April 25, pp. 2598-2606, 1972

Printed in U.S.A.

The Influence of Lipid on the Conformation of Human Plasma High Density Apolipoproteins

(Received for publication, October 13, 1971)

SAMUEL E. Lux, RONALD HIRZ,* RICHARD I. SHRAGER, AND ANTONIO M. GOTTO~

From the Molecular Disease Branch, National Heart and Lung Institute and Division of Computer Research and Technology, National Institutes of Health, Bethesda, Maryland WOO14

SUMMARY

The interaction of human plasma high density apolipopro- teins (apoHDL) with lipids was examined by circular dichro- ism (CD) of the peptide and aromatic chromophores. By CD criteria native HDL contained about 70% LY helical, 5 to 15% /3, and 15 to 20% disordered structure. Delipidation decreased the helical content by about 20% with a corre- sponding increase in disordered structure. The two major apoproteins of HDL (apoA-I and apoA-II), isolated by chro- matography in urea, refolded to different extents after re- moval of urea; apoA-I had more ordered structure than apoA-II (approximately 55 % and 35 % ac helix, respectively). Reconstitution of apoA-I, apoA-II, and apoHDL with both phosphatidylcholine and cholesteryl ester restored, respec- tively, 1 lQ%, 87%, and 100% of the helical structure of the parent HDL. Phosphatidylcholine alone restored 50 to 70 % of the increase produced by both phosphatidylcholine and cholesteryl oleate. With each substrate, the increase in helical content on lipid-protein recombination was accom- panied by a corresponding decrease in disordered structure.

Spectra of HDL in the near-ultraviolet range showed peaks at 258, 264, 283.5, and 290.5 nm. Delipidation reduced and markedly altered the ellipticity bands of the near-ultraviolet CD spectrum. The bands at 283.5 and 290.5 nm, tentatively assigned to one or more of the tryptophan chromophores in apoA-I, were reversed in sign and shifted to 286 and 292 nm, respectively. The near-ultraviolet CD spectrum of the parent HDL was partially restored by recombination of apoHDL with phosphatidylcholine alone and almost com- pletely restored by recombination with both phosphatidyl- choline and cholesteryl oleate. Recombination with phos- phatidylcholine alone was sufficient to restore the 286 and 292 nm peaks to their original sign and position.

These studies indicate that about 20 to 30% of the amino acid residues in the two major HDL apoproteins are involved in a helix-disordered transition on lipid removal or restora- tion. The near-ultraviolet CD spectrum is especially sensi- tive to these lipid-induced structural changes. Both phos- pholipid and cholesteryl esters are required for complete

* Present address, Department of Medicine, Georgetown School of Medicine, Washington, D. C. 20007.

$ Present address, Department of Medicine, Baylor College of Medicine, and The Methodist Hospital, Houston, Texas 77025.

reorganization of the secondary and tertiary structure of HDL.

The nature of the interaction between the lipid and protein moieties of plasma high density lipoproteins is a topic of con- siderable general interest. Previous studies have shown that HDLl contains a relatively high content of ac helix, estimated at 60 to 70%, and that a portion of the helical conformation is lost upon removal of the lipid fraction (l-3). In addition, delipidation substantially diminishes the resistance of the circu- lar dichroism spectrum to change with increasing temperature, suggesting a role for the lipids in stabilizing the conformation of HDL proteins (4).

HDL is a heterogeneous macromolecule containing approxi- mately equal portions of protein and lipid and small amounts of carbohydrate. The protein portion is now known to consist of several components (5-7). Two of these, designated as apoA-I and apoA-II (8) comprise 85 to 90% of the total HDL protein content. ApoA-I is characterized by an amino-terminal aspartic acid and the absence of isoleucine, cysteine, and cystine (5, 8). The carboxyl-terminal amino acid is either threonine (5) or glutamine (8). ApoA-II has a blocked amino terminus, car- boxyl-terminal glutamine (5, 8), and no histidine, arginine, cysteine, or tryptophan. Normally, the ratio of apoA-I and apoA-II is about 3 : 1 (5, 6). The principal lipid components are phospholipids and cholesteryl esters which, together, account for about 85 to 90% of the total HDL lipids (9).

Small amounts of triglyceride and unesterified cholesterol are also present. These lipids may be recombined with HDL pro- teins with a technique devised by Hirz and Scanu (10) employing sonification of the lipid-protein mixture at a temperature above the liquid-crystalline transition of the cholesteryl esters in the lipid mixture (11). HDL reconstituted in this manner closely resembles native HDL in composition, density, thermal stability, and electron microscopic appearance (10, 12).

1 The abbreviations used are: HDL, high density lipoproteins (d = 1.063 to 1.210 g per ml); apoHDL, protein moiety of HDL; apoA-I and apoA-II, the two major apoprotein components of HDL; CD, circular dichroism; ORD, optical rotatory dispersion.

2598

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We have used this recombination technique in the present study to examine the influence of relipidation with phospho- lipid alone or with both phospholipid and cholesteryl ester on the secondary and tertiary structure of HDL apoproteins, as meas- ured by the CD of the peptide and aromatic chromophores. The results indicate that both phospholipid and cholesteryl esters are required to complete the reorganization of the sec- ondary and tertiary structure of HDL.

EXPERIMENTAL PROCEDURB

Materials

Human HDL was isolated from the plasma of normal fasting donors by ultracentrifugal flotation between 1.063 and 1.210 g per ml and washed once by reflotation at 1.210 g per ml (13). The HDL preparations gave a single precipitation line on im- munoelectrophoresis against anti-human whole serum (Hyland) and anti-human HDL. They did not react with antisera to human low density lipoproteins or albumin. Egg yolk phos- phatidylcholine was obtained from Pierce and cholesteryl oleate from Applied Science. Each lipid chromatographed as a single spot on Silica Gel G, developed with chloroform-methanol-water (65:25:4). Urea (Fisher) was deionized by ion exchange chromatography on a mixed resin (Rexyn l-300, Fisher) and was stored at 0” to retard cyanate accumulation (14).

Methods

Preparation of Apoproteins-HDL protein (apoHDL) was prepared by delipidation with ethanol-diethyl ether (1:3) as previously described (3). ApoA-II was obtained by chroma- tography of apoHDL in 6 M urea on DEAE-cellulose as de- scribed by Shore and Shore (5). ApoA-I was obtained by chromatography of apoHDL in 6 M urea on Sephadex G-200 according to the procedure of Scanu et al. (6). Both apoprotein preparations were judged pure (> 98%) on the basis of poly- acrylamide gel electrophoresis in 8 M urea at pH 9.4 and 2.9 and by unique amino acid composition.

Relipidation of Apoproteins-ApoHDL, apoA-I, and apoA-II were combined with phosphatidylcholine alone or phosphatidyl- choline plus cholesteryl oleate using the procedures of Hirz and Scanu (10). Equal weights of each component were used. The recombined high density lipoprotein was isolated by ultra- centrifugal flotation in KBr between densities of 1.063 and 1.210 g per ml.

Protein and Lipid Analyses-The protein concentration was determined in quadruplicate on each preparation by the method of Lowry et al. (15) using bovine serum albumin as a standard. In order to minimize variation, the same micropipette was used for each sample. The variation among the quadruplicate aliquots was %l to 2% (S.D.). Since oxidized lipids may inter- fere with the Lowry procedure (16), in one set of preparations, protein concentrations were also determined by amino acid analysis following a 22-hour hydrolysis in 6 N HCl. There was no difference in the ratio of the protein concentration determined by the two methods when the apoproteins alone were compared with apoproteins recombined with phosphatidylcholine or with apoproteins recombined with both phosphatidylcholine and cholesteryl oleate.

Phosphorus was determined by the method of Chalvardjian and Rudnicki (17). Phospholipid was estimated from phos-

phorus analyses using the factor of 25 g of phospholipid per g of phosphorus. Total cholesterol was determined by a micro- modification of the method of Zak (18).

Circular Dichroism-Circular dichroic spectra were measured in 0.00034 M sodium EDTA, pH 8.0, at room temperature using a Cary 60 spectropolarimeter equipped with a model 6001 CD accessory. The machine was calibrated with d-lo-camphor- sulfonic acid. Potassium dichromate was used to test for ab- sorption artifacts. There was less than 1 mdeg of ellipticity at an absorbance of 2. Determinations were made in 0.5- or l.O- mm cells in the far-ultraviolet region and l.O- and 2.0-cm cells in the near-ultraviolet region. The optical density of all samples was between 0.5 and 1.5 at 225 nm (far-ultraviolet spectra) or 280 nm (near-ultraviolet spectra). Spectra were terminated when the dynode voltage of the sample exceeded the dynode voltage of the blank by 300 volts. Far-ultraviolet CD spectra presented are the mean of three to five separate preparations of HDL, apoHDL, apoA-I, and apoA-II and two to three prepara- tions of relipidated apoproteins. The near-ultraviolet CD spectra are the mean of two sets of preparations. For each preparation, far-ultraviolet spectra were run twice and near- ultraviolet spectra two to four times on the same cell filling. Base-line runs were made as soon before or after each sample run as possible. The signal-to-noise ratio was always greater than 1O:l for the far-ultraviolet spectra and ranged from 4:I to 8: 1 for the near-ultraviolet spectra. The mean residue ellipticity in units of deg cm2 dmole-l was calculated from:

[eh = (MRW)e”x

1ozc

where 0”~ is the observed ellipticity in degrees at wave length X, 1 is the optical path in centimeters, e is the concentration in grams per ml, and MRW is the mean residue weight. Values of MRW were calculated from amino acid analyses of the samples used and were 112.1 (apoA-II), 116.6 (apoA-I), and 114.7 (apoHDL).

Analysis of Circular Dichroic Spectra-The far-ultraviolet spectra were analyzed between 208 and 240 nm with a computer graphics program (Modelaide). The program utilized a tech- nique of nonlinear regression with linear constraints (19) to obtain the best fit of the unknown spectra and model spectra in the a! helical, p, and disordered conformation.2 Even the poorest fits were quite good. In all cases the average difference between the computed and measured ellipticities was less than 7% of the average measured ellipticity. The computed fractions of a! helical, p, and disordered conformation were constrained so that they were greater than or equal to 0, and the sum of the three fractions was 1. Three sets of model spectra were em- ployed.

ikfoclel I-Spectra of poly-L-lysine solutions in the a: helical, & and disordered conformations were used as suggested by Greenfield and Fasman (20) employing the values determined by these investigators.

Model II-As in Model I, spectra of poly-n-lysine solutions were used to define a! helical and p conformations. The spec-

2 In this paper disordered conformation refers to the nonregular, nonrepeating structures in proteins. The terms unordered struc- ture, random structure, and random coil are frequently used synonomously.

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2600 Lipid-Protein Interactions in HDL Vol. 247, No. 8

TABLE I Composition of reconstituted lipoproteins

HDL apoproteins were recombined with either phosphatidyl- choline alone or phosphatidylcholine and cholesteryl oleate. Equal weights of each protein and lipid were used. The recom- bined lipoprotein complex was isolated between densities 1.063 to 1.210 g per ml and analyzed for protein, lipid, and total choles- terol. The results presented are from a single set of preparations.

Compound’

HDLb apoHDL apoHDL + PC apoHDL + PC + CO apoA-I apoA-I + PCC apoA-I + PC + CO apoA-II apoA-II + PCc apoA-II + PC + CO

Composition

Protein

48 99 51 46 99 43 42 99 59 40

Phospholipid Total cholesterol

weight 7%

27 <0.4 49 38

<0.8 57 48

<0.2 41 38

21 <0.7

15 <0.4

10 <0.2

21

a The abbreviat.ions used are: PC, phosphatidylcholine; CO, cholesteryl oleate.

b The composition of HDL does not include the content of tri- glyceride (4yo).

c Total cholesterol was not determined on these fractions.

+I0

+5

0

u

E s -5

:

g ‘0 -10

n ‘0 x -15

z

-20

-25

-30

- HDL - -- apoHDL -.-.- apoA-I . . . . . . . . . apoA.n

1 I I I I - 200 210 220 230 240

WAVELENGTH (nm) 25(

FIG. 1. The far-ultraviolet CD spectra of HDL, apoHDL, apo- A-I, and apoA-II.

trum of a film of poly-r,-lysine in the disordered form was sub- stituted for the spectrum of the disordered form of poly-n-lysine in solution as suggested by Fasman, Hoving, and Timasheff (21). Values for the poly-L-lysine film were obtained from Fig. 6 of Reference 21.

Model III-The spectral parameters calculated by Saxena and Wetlaufer were used (22). They computed model spectra for cx helical, /I, and disordered conformations from the CD spectra of myoglobin, lysozyme, and ribonuclease and the content of (Y helical, p, and disordered structure in these three proteins, determined from x-ray diffraction analysis.

The curve-fitting program was tested on myoglobin, ribo- nuclease, and lysozyme, using CD spectra of each protein com- piled by Saxena and Wetlaufer (22). As expected, using the Model I and Model III systems, the calculated percentages of a! helix, /?, and disordered structure in each of the three pro- teins were within 1% of the percentages previously derived (20, 22) using other curve-fitting procedures and within 50/, of the mean conformation as determined by x-ray diffraction. Use of a disordered film of poly-L-lysine to simulate the disordered chains of proteins, as in the Model II system, has not previously been reported, although several authors (21, 23) have suggested that such films are more reasonable models for disordered protein structures than disordered polypeptides in solution. The Model II system gave satisfactory estimations of (Y helical content and correctly predicted the content of /I and disordered structure in myoglobin. However, in lysozyme and ribonuclease Model II underestimated the percentage of fl structure and overestimated the percentage of disordered structure by 10 to 25%.

RESULTS

Composition of Reconstituted Lipoproteins-The composition of protein, phospholipid, and cholesterol for one set of preparations is presented in Table I. No lipid is detectable in the apoprotein preparations. The figures shown for the apoproteins represent the smallest amounts that could have been detected under the assay conditions. The composition of the apoproteins recom- bined with both phosphatidylcholine and cholesteryl oleate differs somewhat from the composition of the same apoproteins relipidated with whole HDL lipids, previously reported by Scanu et al. (12). In the latter study the protein content was 5 to 8% higher, the phospholipid content 5 to 18% lower, and the choles- teryl ester content approximately the same as in the present study. Presumably these variations reflect differences in the composition of the lipids used for relipidation, although varia- tions of nearly this magnitude have been reported for the com- position of HDL analyzed in different laboratories (9, 24).

Far-Ultraviolet Circular Dichroism of HDL and HDL Apo- proteins-The far-ultraviolet CD spectra of HDL and apoHDL (Fig. 1) display the deep double troughs at 208 and 221 nm corresponding to the ?r + r* (parallel) and n + rr* transitions of the peptide bonds in helical polypeptides (25). They are comparable to HDL and apoHDL spectra previously published by Gotto and Kon (3) and Scanu and Hirz (2). Spectral analysis (Table II) indicates that HDL contains about 70% (Y helix, 5 to 15% fi structure, and 15 to 20% disordered structure. Delipidation decreases the helical content to about 50yo with a corresponding increase in disordered structure. These spectral alterations can probably be attributed entirely to changes in protein conformation since the extracted lipids in hexane have no detectable optical activity between 200 and 250 nm.

In agreement with previous reports (6, 26), apoA-I has more ordered structure than apoA-II (Fig. 1). The computed values for the LY helical content of apoA-I (54 to 57%) and apoA-II (33 to 38%) are in good agreement with the estimations of 48 to 51% and 32 to 33%,, respectively, previously reported by

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TABLE II

2601

Computed conformations of HDL, apoproteins, and reconstituted lipoproteins

Model Ib Model IIb Model III6

RMSC h&c

-

B RMS= h& Dis-

rdered

% 20

d “Eo2 % % % , ‘e&y %

(418) 72 6 22 (769 ) 70

Mean -

B 01

-

%

11

%

19

45 (498 1 54 11 35 (811) 52 10 38 32 (553 ) 66 5 29 (961) 64 8 28 23 (465 1 72 4 24 (784) 70 8 22

43 (572) 57 9 34 (887 ) 55 8 37 23 (625 ) 72 4 24 (1180) 69 9 22 16 (813) 83 0 17 (1430) 83 2 15

63 (324) 38 17 45 (427 ) 35 46 (366) 52 12 36 (405 ) 50 39 (488 ) 63 6 31 (878) 61

13 11

6 -

52 39 33

- _

httk s Disor- dered RMSC hzix B

% % %

69 16 15

d 3n%*’ %

(358) 69

%

11

51 15 34 (505 ) 50 5 63 13 24 (500 ) 62 6 69 13 18 (396 ) 69 8

55 12 33 (566) 54 3 68 14 18 (553 1 68 9 84 4 12 (665 ) 83 1

35 17 48 (341) 33 4 49 16 35 (284 ) 48 6 60 10 30 (470 ) 60 1

- 1

-

HDL

apoHDL apoHDL + PC apoHDL + PC + CO

apoA-I apoA-I + PC apoA-I + PC + CO

apoA-II apoA-II + PC apoA-II + PC + CO

a The abbreviations used are: PC, phosphatidylcholine; CO, cholesteryl oleate. b Model I utilized the spectra of poly-L-lysine solutions in the (Y helical, p, and disordered conformations. Model II employed the

spectra of poly-n-lysine solutions in the a helical and 0 conformations and the spectrum of a poly-n-lysine film in the disordered con- formation. Model III utilized spectra for the (Y helical, 0, and disordered conformations computed by Saxena and Wetlaufer (22) from the CD spectra of myoglobin, lysozyme, and ribonuclease and the known structure of these three proteins as determined by x-ray diffraction analysis.

c RMS refers to the root mean sauare of the difference in ellinticities between the observed and computed curves. This is a measure of the closeness of the fit procedure.

Gotto and Shore (26). In addition, the spectral analysis sug- gests that both apoA-I and apoA-II contain small amounts of /3 structure, perhaps 5 to 15% (Table II). Note that even the Model II system, which underestimated the p structure in two of three proteins of known structure predicts some p structure in these apoproteins. Since these apoproteins are isolated by chromatography in 6 M urea and since urea in this concentration has been shown to denature apoHDL (27), an important con- sideration is whether or not the denaturation is reversible. Three observations suggest that the refolding of each apoprotein is essentially complete. First, the effects of 6 M urea on the CD spectrum of apoHDL are completely reversed by dialysis. Second, as noted above, previous studies have shown that together apoA-I and apoA-II constitute 85 to 90% of apoHDL and that normally they are present in a ratio of 3 : 1 (apoA-I : apoA- II) (6, 7). A linear recombination, between 208 and 240 nm, of three parts of the apoA-I spectrum and one part of the apoA-II spectrum closely simulates the spectrum of apoHDL. Third, when native HDL is dehydrated and rehydrated, apoA-I is dissociated from its lipid complement and may be isolated free of the other HDL apoproteins by ultracentrifugation at 1.210 g per ml (28). This lipid-free apoA-I has never been exposed to urea or the organic solvents used in delipidation, but its far- ultraviolet CD spectrum is identical with apoA-I isolated by the conventional procedure.

Far-Ultraviolet Circular Dichroism of Relipidated HDL Apo- proteins-The far-ultraviolet CD spectra of apoHDL recombined with phosphatidylcholine alone or with phosphatidylcholine and cholesteryl oleate are presented in Fig. 2. The spectrum of apoHDL recombined with both lipids is indistinguishable from the spectrum of native HDL, shown as a shaded area representing t,he range of five separate HDL preparations. Reconstitution

-301 I I I I I 200 210 220 230 240 2

WAVELENGTH (nm) '0

FIG. 2. The far-ultraviolet CD spectra of apoHDL, apoHDL recombined with phosphatidylcholine alone (apoHDL + PC), and apoHDL recombined w-ith both phosphatidylcholine and cholesteryl oleate (apoHDL + PC + CO). The shaded area rep- resents the range of the CD spectra of five separate HDL prepara- tions.

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2602 Lipid-Protein Interactions in HDL

- apoA-II

i --- apoA-II+PC

\ --------- apoA-II+PC+CO

-xJ I I I I I I 200 210 220 230 240 250

WAVELENGTH (nm)

B

- apoA-I

, --- apoA-I +PC ------ apoA-I+PC+CO

I I I I I

200 210 220 230 240 : WAVELENGTH hm)

Vol. 247, No. 8

FIG. 3. The far-ultraviolet CD spectra of (A) apoA-II, apoA-II recombined with phosphatidylcholine (apoA-ZZ + PC), and apo- A-II recombined with both phosphaGdylcholine and cholesteryl oleate (apoA-ZZ + PC + CO), and (B) apoA-I, apoA-I + PC, and apo A-I + PC + CO. The shaded area represents the range of five separate HDL preparations.

+50 I I I I I I

286 292

258 264 283.5 290:5 296

+25 I I I III

-75u I I 1 I I I 250 260 270 280 290 300 310 320

WAVELENGTH (nm)

FIG. 4. The near-ultraviolet CD spectra of HDL apoHDL, apo- A-I, and apoA-II.

with phosphatidylcholine alone restores 610/, of the increase in [0]221 produced by both lipids. Sonification of HDL under the conditions used to effect reconstitution produces no change in its far-ultraviolet CD spectrum. The spectral analysis (Table II) suggests that the increase in helical content on relipidation with either phospholipid or phospholipid and cholesteryl ester is almost completely compensated by a decrease in disordered structure and that little or no net change in /3 structure occurs on relipidation.

The effects of recombination with phosphatidylcholine alone or phosphatidylcholine plus cholesteryl oleate on the CD spectra of apoA-II (Fig. 3A) and apoA-I (Fig. 3B) are analogous to their

effects on apoHDL. There is an increase in the magnitude of the negative ellipticity of both apoproteins consequent to re- combination with phosphatidylcholine and an even greater increase when cholesteryl oleate is also recombined. At each stage of relipidation, the apoA-I lipoprotein complex displays more ordered structure than the corresponding apoA-II complex; however, the relative change induced by phosphatidylcholine alone as compared to phosphatidylcholine plus cholesteryl oleate is approximately the same for each apoprotein. For example, reconstitution of apoA-I with phosphatidylcholine alone restores 59% of the increase in [0]221 and 44% of the in- crease in [0],, produced by relipidation with both lipids. The corresponding figures for apoA-II are 61% and 51%. The spectral analysis (Table II) suggests that both apoA-I and apoA-II show a 10 to 150/, increase in a! helical content, a 10 to 20% decrease in disordered structure, and little or no net change in the content of /3 structure on relipidation with phos- phatidylcholine alone and a 25 to 30% increase in a! helical content, a 15 to 30% decrease in disordered structure, and a 0 to 10% decrease in /3 structure on relipidation with both phos- phatidylcholine and cholesteryl oleate.

One important question that arises is whether the conforma- tion of these isolated, relipidated apoproteins approximates their conformation in native HDL or not. While no conclusive proof is possible, two lines of evidence suggest that such an extrapola- tion may be at least qualitatively applicable. First, the ampli- tude of the spectra of apoA-II and apoA-I relipidated with both phospholipid and cholesteryl ester are of the right order of magnitude; the apoA-II complex contains less ordered structure and the apoA-I complex contains more ordered structure than the parent HDL (Fig. 3). A linear recombination of appropriate proportions of the spectrum of each complex approximates the spectrum of the parent HDL. Second, as noted above, when HDL is dehydrated and rehydrated, apoA-I is dissociated from

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Issue of April 25, 1972 X. E. Lux, R. Him, R. I. Xhrager, and A. M. Gotto 2603

its lipid complement and sediments at a density of 1.210. The high density lipoprotein fraction (d 1.063 to 1.210) remaining contains 3570 protein, 467, phospholipid, 5% cholesterol, 11 7. cholesteryl ester, and 3% triglyceride (28) ; 85 to 90% of the protein is apoA-II (28). Although this apoA-II-rich lipo- protein fraction has been produced in an entirely different man- ner from the HDL complex obtained on relipidation of apoA-II with phosphatidylcholine and cholesteryl oleate, the two CD spectra are very similar. So far no comparable means for isolating a high density lipoprotein fraction containing only apoA-I has been discovered.

Near-Ultraviolet Circular Dichroism of HDL and HDL Apo- proteins-The CD spectrum of HDL in the near-ultraviolet, between 250 and 315 nm, displays positive extrema at 258, 264, 283.5, and 290.5 nm, and negative extrema at 262, 269, 286, and 296 nm (Fig. 4). Delipidation markedly reduces the over-all ellipticity of the near-ultraviolet CD spectrum. The positive extrema at 258 and 264 nm remain. The positive extrema at 283.5 and 290.5 nm are replaced by a negative extremum at 292 nm with a shoulder at approximately 286 nm. The negative

TABLE III

Content of aromatic amino acids and half-cystine in apoA-I and apoA-II

Data from Shore and Shpre (29). Assumed residues per mole- cule were calculated using the molecular weights of 14,900 (apoA- II) and 15,500 (apoA-I) determined by Shore and Shore.

Amino acid ApoA-I I

ApoA-II

assnnzed residues/molectrle

Tyrosine 4 6 Tryptophan 4 0 Phenylalanine 3 7 Half-cystine. 0 2

extremum at 296 nm is no longer clearly identifiable. A broad positive plateau of low ellipticity replaces the negative extremum centered at 268 nm. These spectral changes can probably be attributed entirely to changes in protein conformation since the extracted lipids, in hexane or ethanol, have no optical activity between 250 and 320 nm.

The near-ultraviolet CD spectra of apoA-I and apoA-II (Fig. 4) provide some insight into the origin of these CD features. ApoA-II, which contains no tryptophan (Table III), is missing the 296, 292, and 286 nm extrema. ApoA-I, which contains no cystine or cysteine, displays extrema at 258, 262, 264, 268, and 292 nm and a shoulder at approximately 286 nm.

On titration of HDL from pH 9.55 to 12.60 (Fig. 5), there is a general trend for increasingly positive ellipticity values as the pH becomes more alkaline. At pH 10.72, some fine structure appears in the 270 to 280 nm band which disappears again at higher pH values. Circular dichroic difference spectra, ob- tained by subtracting the CD spectrum at pH 9.55 from the spectra at higher pH values (Fig. 5, inset), indicate that the maximum changes in ellipticity occur in a band at or below 250 nm, in the broad 270 to 280 nm region, and in a band centered at 295 nm. This is compatible with the interpretation that at least a portion of the ellipticity represented in the 270 to 280 nm region is contributed by tyrosine residues. Titration of these residues is accompanied by a change in ellipticity at 270 to 280 nm and the appearance of a new ellipticity band centered at 295 nm, an absorption maximum of the tyrosinate ion.

As with the far-ultraviolet CD spectra, a linear recombination of three parts of the apoA-I and one part of the apoA-II spectra between 250 and 315 nm simulates the apoHDL spectrum within experimental error, providing additional evidence that the refolding apoA-I and apoA-II is essentially complete.

The near-ultraviolet CD spectrum of HDL is partially (50 to 70%) restored by recombination of apoHDL with phosphat.idyl-

,h. PH

- 9.55 -.-.- 10.42 _____ _ _.-. 10.72 ---- Il.74 __-..- ,2 60

a z E

NY 5

F 2

E

250 260 270 280 290 300 310 320

WAVELENGTH (nm’)

FIG. 5. The near-ultraviolet CD spectra of HDL as a function were obtained after aliquots of a single HDL preparation were of pH from pH 9.55 to 12.60. The near-ult,raviolet CD difference equilibrated for 24 hrs in glycine-NaOH (pH 9.66 and iO.&), spectra, obtained by subtracting the CD spectrum at pH 9.55 from NazHP04-NaOH (pH 10.72 and 11.74), or NaOH-NaCl (pH 12.60) the spectra at higher pH, are shown in the inset figure. Spectra buffers. The ionic strength of all the buffers was 0.1.

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2604 Lipid-Protein Intel-actions in HDL Vol. 247, No. 8

258 264 283.5 290.5 I I I I

+25

-.-.- HDL

- opoHDL --- apoHDL+ PC .------ opoHDL t PC tC0

-100 I I I I , , 250 260 270 280 290 300 310 320

WAVELENGTH hm)

FIG. 6. The near-ultraviolet CD spectra of HDL, apoHDL, apoHDL recombined with phosphatidylcholine alone (apoHDL + PC) and apoHDL recombined with both phosphatidylcholine and cholesteryl oleate (apoHDL + PC + CO).

choline alone and almost completely restored by recombination with both phosphatidylcholine and cholesteryl oleate (Fig. 6). Note that recombination with phosphatidylcholine alone is sufficient to restore the 283.5 and 290.5 nm extrema (which were inverted and red-shifted to 286 and 292 nm in apoHDL) to their original sign and position. The minor differences between the recombined apoHDL and t.he parent HDL spectra are within experimental error (signal-to-noise ratio of 4: 1 to 5:l from 250 to 285 nm) except at the 290.5 nm peak (signal-to- noise ratio of 8: 1) which was not completely restored in the reconstituted complex.

DISCUSSION

These studies indicate that the interaction of lipids and pro- teins in apoHDL and its major protein subunits, apoA-I and apoA-II, is accompanied by a reversible conformational change. This change affects both the tertiary structure, as monitored by near-ultraviolet CD spectra, and the secondary structure, as monitored by far-ultraviolet CD spectra. These two spectral regions will be discussed separately.

Near-Ultraviolet Region-Studies of the CD of proteins in the near-ultraviolet region have been hampered by the low spectral intensities observed for most proteins and by difficulties in interpreting features of the complex spectra. The latter problem has been considerably alleviated by the studies of Edelhoch, Horwitz, Strickland, and their colleagues using diketopiperazine model compounds and low temperature (77” K) spectroscopy (30-35) to resolve the fine structure in near-ultraviolet CD spectra of aromatic chromophores. Nevertheless, it is often still difficult to make definitive assignments, especially in pro- teins containing multiple chromophores in varying environments.

In HDL (Fig. 4) the extrema at 283.5 and 290.5 nm can probably be assigned to one or more of the tryptophanyl residues of apoA-I on the basis of their location, like sign, and characteris- tic spacing (30,32,35). These bands correspond to two different vibrational states of the ‘La electronic transition of tryptophan (30, 32, 351 Simihr features have been observed in a variety

of proteins including bovine growth hormone (31), porcine thyrocalcitonin (36)) egg white lysozyme (37), P-lactoglobulin (38), and concanavalin A (39). As expected, apoA-II, which contains no tryptophan, is lacking this transition (Fig. 4). In tryptophanyl model compounds the location of these bands varies with the solvent environment (30). Following delipida- tion of HDL, the 283.5 and 290.5 nm bands are reversed in sign and shifted to 286 and 292 nm, respectively, indicating that the tryptophan residues contributing to the CD spectrum have assumed a new average conformation and are in a more nonpolar environment (Fig. 4). It is of interest that the interaction of apoHDL with phospholipid alone is sufficient to restore these residues to their original conformation and environment (Fig. 6). The addition of cholesteryl ester intensifies these bands, probably by further stabilizing this conformation but does not alter their sign or position. This is compatible with a previous hypothesis (40) that the primary lipid-protein interaction in HDL is between proteins and phospholipids.

On the basis of studies with diketopiperazines (31) and model compounds of phenylalanine (33), the extrema at 258 and 264 nm in HDL (Fig. 4) are likely due to phenylalaninyl residues. Disulfide bonds can also contribute in this spectral region (41), but they show no fine structure (34). In addition, apoA-I which contains no cysteine or cystine, prominently displays these bands (Fig. 4). Unlike the bands assigned to tryptophans, the phenylalaninyl bands show little change on delipidation; however, at least a portion of the CD originating from phenyl- alanine is due to the intrinsic dyssymmetry of the residues them- selves and is not dependent on tertiary interactions (42). Fur- ther studies will be required to assess the proportion of these bands which are responsive to conformational change.

At the present time, it is not possible to assign the residues responsible for the CD contributions in the 270 to 280 nm region with any confidence. Tryptophan, tyrosine, and disulfides may all contribute in this spectral range. As noted under “Results,” the spectral changes accompanying titration of HDL from pH 9.55 to 12.60 suggest that at least a portion of the ellipticity in this region is contributed by tyrosinyl residues. However, since the major CD bands in tyrosinyl model compounds occur be- tween 275 and 285 nm (30, 32, 35) and since the negative ex- tremum is centered at 268 to 270 nm, contributions of other residues to this band are likely. In particular, transitions resembling the strong broad negative HDL band centered at 268 to 270 and the weak negative HDL band centered at 296 nm have been observed in tryptophanyl model compounds and attributed to IL, transitions (35). In addition, the possibility that the single disulfide bond in apoA-II (Table III) may also contribute to the 270 to 280 band cannot be be ruled out.

Far- Ultraviolet Region-The interaction between lipids and proteins in HDL is accompanied by substantial changes in the secondary structure of the protein. Delipidation of HDL re- duces the ellipticity of the far ultraviolet CD spectrum by 20 to 30%. About 60% of this decrease is restored by relipidation with phosphatidylcholine alone, while relipidation with both phosphatidylcholine and cholesteryl oleate completely restores the parent HDL spectrum. This is in agreement with prelim- inary results reported by Hirz and Scanu in which relipidation of apoHDL with the lipids of whole HDL completely restored the native HDL spectrum (10). Both apoA-I and apoA-II show changes on relipidation similar to those observed for apo- HDL.

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Issue of April 25, 1972 X. E. Lxx, R. Hirx, R. I. Shrager, and A. M. Gotto 2605

In order to determine the specific nature of these conformational changes, an effort was made to estimate the proportions of cy helical, p, and disordered structure present in each preparation using various model spectra. The theoretical and practical limitations of this approach have been extensively reviewed (20, 22, 23, 43). The technique is undoubtedly an oversimplifica- t.ion and at the present stage of understanding, the computed results must be considered only an approximation of the actual conformation in solution. Potential errors arise from (a) the differences between the structure of disordered peptides in solution and the constrained disordered chains of proteins (21, 23, 44), (b) residues in conformations other than the funda- mental three employed, (c) variations in the micropolarity about individual chromophores (43), (d) chain length effects (45, 46), and (e) contributions from side chain chromophores (23, 25, 47, 48). In addition, quantitative and qualitative variations in the spectra of various synthetic polypeptides which are thought to be in the same conformation raise doubts about the choice of an appropriate reference system (49-52). Despite these theo- retical limitations, the method has proved accurate in predicting secondary structure in a number of proteins and in all cases has equalled or surpassed predictions obtained by other methods of ORD or CD analysis (20, 22). In the present paper, three reference systems were employed. Model I has been found to be particularly useful in predicting the secondary structure of proteins containing considerable or helix or p structure (e.g. myoglobin, lysozyme, and ribonuclease) but was less accurate when applied to proteins containing largely disordered structure (e.g. carboxypeptidase A) (20). Model III, derived from the CD spectra of myoglobin, lysozyme, and ribonuclease, has the advantage that alterations induced by variations in the environ- ment of the peptide bonds and the chain length of Q! helical or /3 structured segments in these three proteins are incorporated into the model (22). Model II was constructed on the premise that films of synthetic polypeptides in the disordered form might represent the disordered constrained polypeptide chains of proteins better than disordered polypeptides in solution (21). This model underestimated the content of /3 structure and over- estimated the content of disordered structure in ribonuclease and lysozyme, but worked well with myoglobin, a protein con- taining no /3 structure.

The computed conformations of HDL, the apoproteins, and the lipid-protein complexes obtained with the different model systems agree surprisingly well. This suggests that the com- puted conformations may be reasonably accurate. The analysis indicates that 01 helical and disordered structures predominate in this group of proteins. On the average, small amounts (2 to 13%) of /I structure are predicted for each of the preparations. At present, it is difficult to assess the accuracy of this prediction. Infrared spectra of the amide I band of HDL and apoHDL (3) do not show any of the features of p structure, although an in- tensive search for these features using highly concentrated protein solutions has not been made. In addition, it is not certain that infrared methods are sufficiently sensitive to detect the presence of ,8 structure in such small proportions. Analysis by each of the three methods suggests that the conformational change on delipidation of HDL or relipidation of apoHDL, apoA-I, or apoA-II, is principally a helix-disordered transition with little or no net change in the predicted content of /I struc- ture. This transition involves approximately 20 to 30% of the amino acid residues. If, as reported, both apoA-I and

apoh-II have molecular weights of 14,000 to 16,000 (29), the transition probably involves between 25 and 40 residues in each protein.

Although it is by no means clear how the lipid moieties mediate their influence on the protein folding, it is possible that analogies exist between their ability to increase the proportion of helical conformation and the well known ability of anionic detergents (53, 54) and certain organic solvents such as 2- chloroethanol (55, 56) to increase helical content. According to this hypothesis, the amino acid residues in HDL involved in the helix-disordered transition would have to be at or near the lipid-binding site or sites. The binding of lipids would decrease the local polarity at these sites and produce a nonpolar, weakly hydrogen-bonding enviroument which would favor the formation of increased helical structure, a conformation in which the peptide groups would be hydrogen-bonded to each other and shielded from the nonpolar environment. Delipidation would be expected to expose the residues at the lipid-binding site and favor a transition to a more disordered structure in which the peptide bonds could interact maximally with the aqueous solvent. It is hoped that current efforts to isolate and char- acterize lipid-binding fragments from HDL apoproteins will allow a more accurate assessment of this hypothesis.

The present studies indicate that both phospholipids and cholesteryl esters are required for complete reorganization of the secondary and tertiary structure of HDL. This agrees with recent studies from this laboratory in which the relipidation of spin-labeled apoHDL w-as monitored by electron spin resonance spectroscopy (3, 57). In native HDL signals from both strongly and weakly immobilized spin labels were evident. The strongly immobilized component of the electron spin resonance spectrum of native HDL was lost on delipidation and was only restored when apoHDL was reconstituted with both phosphatidylcholine and a nonpolar lipid (cholesteryl oleate or triolein). The ob- servation that the electron spin resonance spectrum and the near- and far-ultraviolet CD spectra are all regenerated in the reconstituted HDL strongly suggests that the reconstitution procedure is highly effective in restoring the native conformation of the HDL protein components.

Acknowledgments-We wish to thank Ms. Kathryn John for assistance in preparation of the lipoprotein and apoprotein fractions, Dr. Harold Edelhoch for critical and stimulating discussions, and Drs. V. 1’. Saxena and D. B. Wetlaufer for al- lowing us to read their manuscript prior to publication.

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3. 4. 5. G.

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Supplement III, III-S

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Samuel E. Lux, Ronald Hirz, Richard I. Shrager and Antonio M. GottoApolipoproteins

The Influence of Lipid on the Conformation of Human Plasma High Density

1972, 247:2598-2606.J. Biol. Chem. 

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