Direct Measurement of the Structure of Reconstituted High-Density Lipoproteins by Cryo-EM

Early forms of high-density lipoproteins (HDL), nascent HDL, are formed by the interaction of apolipoprotein AI with macrophage and hepatic ATP-binding cassette transporter member 1. Various plasma activities convert nascent to mature HDL, comprising phosphatidylcholine (PC) and cholesterol, which are selectively removed by hepatic receptors. This process is important in reducing the cholesterol burden of arterial wall macrophages, an important cell type in all stages of atherosclerosis. Interaction of apolipoprotein AI with dimyristoyl (DM)PC forms reconstituted (r)HDL, which is a good model of nascent HDL. rHDL have been used as an antiathersclerosis therapy that enhances reverse cholesterol transport in humans and animal models. Thus, identification of the structure of rHDL would inform about that of nascent HDL and how rHDL improves reverse cholesterol transport in an atheroprotective way. Early studies of rHDL suggested a discoidal structure, which included pairs of antiparallel helices of apolipoprotein AI circumscribing a phospholipid bilayer. Another rHDL model based on small angle neutron scattering supported a double superhelical structure. Herein, we report a cryo-electron microscopy-based model of a large rHDL formed spontaneously from apolipoprotein AI, cholesterol, and excess DMPC and isolated to near homogeneity. After reconstruction we obtained an rHDL structure comprising DMPC, cholesterol, and apolipoprotein AI (423:74:1 mol/mol) forming a discoidal particle 360 Å in diameter and 45 Å thick; these dimensions are consistent with the stoichiometry of the particles. Given that cryo-electron microscopy directly observes projections of individual rHDL particles in different orientations, we can unambiguously state that rHDL particles are protein bounded discoidal bilayers.     

Cholesterol is a determinant of the structures of discoidal high density lipoproteins formed by the solubilization of phospholipid membranes by apolipoprotein A-I

Formation of discoidal high density lipoproteins (rHDL) by apolipoprotein A-I (apoA-I) mediated solubilization of dimyristoyl phosphatidylcholine (DMPC) multilamellar vesicles (MLV) was dramatically affected by bilayer cholesterol concentration. At a low ratio of DMPC/apoA-I (2 mg DMPC/mg apoA-I, 84/1 mol/mol), sterols (cholesterol, lathosterol, and beta-sitosterol) that form ordered lipid phases increase the rate of solubilization similarly, yielding rHDL with similar structures. By changing the temperature and sterol concentration, the rates of solubilization varied almost 3 orders of magnitude; however, the sizes of the rHDL were independent of the rate of their formation and dependent upon the bilayer sterol concentration. At a high ratio of DMPC/apoA-I (10/1 mg DMPC/mg apoA-I, 420/1 mol/mol), changing the temperature and cholesterol concentration yielded rHDL that varied greatly in size, phospholipid/protein ratio, mol% cholesterol, and number of apoA-I molecules per particle. rHDL were isolated that had 2, 4, 6, and 8 molecules of apoA-I per particle, mean diameters of 117, 200, 303, and 396 A, and a mol% cholesterol that was similar to the original MLV. Kinetic studies demonstrated that the different sized rHDL are formed independently and concurrently. The rate of formation, lipid composition, and three-dimensional structures of cholesterol-rich rHDL is dictated primarily by the original membrane phase properties and cholesterol content. The size speciation of rHDL and probably nascent HDL formed via the activity of the ABCA1 lipid transporter is mechanistically linked to the cholesterol content of the membranes from which they were formed.     

Incorporation of cholesterol into serum high density lipoprotein apoprotein and recombinants

The uptake of cholesterol (CHL) by serum high density lipoprotein (HDL) delipidated apoproteins and phospholipid-apoprotein recombinants has been studied with two methods; by incubation with Celite-dispersed cholesterol or with cholesterol crystals. The apoproteins bind very small amounts of cholesterol with a maximum of about 6 micrograms/mg apoprotein. Recombinants with dimyristoyl phosphatidylcholine (DMPC) or egg phosphatidylcholine (EPC) as phospholipid component gave similar values for cholesterol uptake. The initial rate for uptake from Celite-cholesterol by recombinants was high (0.1 mol cholesterol/mol phospholipid/h) and somewhat higher than that for phospholipid vesicles. The maximal uptake was by gel filtration shown to depend on the size of the complexes with values about 0.95 mol cholesterol per phospholipid for vesicular complexes, 0.75 for discoidal complexes and between 0.5 and 0.2 for small 'protein-rich' complexes. During the incubation of recombinants with cholesterol there was considerable decomposition of discoidal complexes and formation of larger ones. The results show that phospholipid-apoprotein complexes are efficient acceptors for cholesterol but also that about 25% of the phospholipid in the discoidal complexes is excluded from interaction with cholesterol by interaction with apoprotein.     

Interaction of cholesterol,phospholipid and apoprotein in high density lipoprotein recombinants

To examine the effect of incorporation of cholesterol into high density lipoprotein (HDL) recombinants, multilamellar liposomes of ³H cholesterol/¹⁴C dimyristoyl phosphatidylcholine were incubated with the total apoprotein (apoHDL) and principal apoproteins (apoA-1 and apoA-2) of human plasma high density lipoprotein. Soluble recombinants were separated from unreacted liposomes by centrifugation and examined by differential scanning calorimetry and negative stain electron microscopy. At 27°C, liposomes containing up to approx. 0.1 mol cholesterol/mol dimyristoyl phosphatidylcholine (DMPC) were readily solubilized by apoHDL, apoA-1 or apoA-2. However, the incorporation of DMPC and apoprotein into lipoprotein complexes was markedly reduced when liposomes containing a higher proportion of cholesterol were used. For recombinants prepared from apoHDL, apoA-1 or apoA-2, the equilibrium cholesterol content of complexes was approx. 45% that of the unreacted liposomes. Electron microscopy showed that for all cholesterol concentrations, HDL recombinants were predominantly lipid bilayer discs, approx. $\text{160 × 55}\text{A}\text{?}$. Differential scanning calorimetry of cholesterol containing recombinants of DMPC/cholesterol/apoHDL or DMPC/cholesterol/apoA-1 showed, with increasing cholesterol content, a linear decrease in the enthalpy of the DMPC gel to liquid crystalline transition, extrapolating to zero enthalpy at 0.15 cholesterol/DMPC. The enthalpy values were markedly reduced compared to control liposomes, where the phospholipid transition extrapolated to zero enthalpy at approx. 0.45 cholesterol/DMPC. The calorimetric and solubility studies suggest that in high density lipoprotein recombinants cholesterol is excluded from 55% of DMPC molecules bound in a non-melting state by apoprotein.     

Interaction of cholesterol, phospholipid and apoprotein in high density lipoprotein recombinants.

To examine the effect of incorporation of cholesterol into high density lipoprotein (HDL) recombinants, multilamellar liposomes of 3H cholesterol/14C dimyristoyl phosphatidylcholine were incubated with the total apoprotein (apoHDL) and principal apoproteins (apoA-1 and apoA-2) of human plasma high density lipoprotein. Soluble recombinants were separated from unreacted liposomes by centrifugation and examined by differential scanning calorimetry and negative stain electron microscopy. At 27 degrees C, liposomes containing up to approx. 0.1 mol cholesterol/mol dimyristoyl phosphatidylcholine (DMPC) were readily solubilized by apoHDL, apoA-1 or apoA-2. However, the incorporation of DMPC and apoprotein into lipoprotein complexes was markedly reduced when liposomes containing a higher proportion of cholesterol were used. For recombinants prepared from apoHDL, apoA-1, or apoA-2, the equilibrium cholesterol content of complexes was approx. 45% that of the unreacted liposomes. Electron microscopy showed that for all cholesterol concentrations, HDL recombinants were predominantly lipid bilayer discs, approx. 160 X 55 A. Differential scanning calorimetry of cholesterol containing recombinants of DMPC/cholesterol/apoHDL or DMPC/cholesterol/apoA-1 showed, with increasing cholesterol content, a linear decrease in the enthalpy of the DMPC gel to liquid crystalline transition, extrapolating to zero enthalpy at 0.15 cholesterol/DMPC. The enthalpy values were markedly reduced compared to control liposomes, where the phospholipid transition extrapolated to zero enthalpy at approx. 0.45 cholesterol/DMPC. The calorimetric and solubility studies suggest that in high density lipoprotein recombinants cholesterol is excluded from 55% of DMPC molecules bound in a non-melting state by apoprotein.     

Electrostatic effects on the stability of discoidal high-density lipoproteins

High-density lipoproteins (HDL) remove cholesterol from peripheral tissues and thereby help to prevent atherosclerosis. Nascent HDL are discoidal complexes composed of a phospholipid bilayer surrounded by protein alpha-helices that are thought to form extensive stabilizing interhelical salt bridges. Earlier we showed that HDL stability, which is necessary for HDL functions, is modulated by kinetic barriers. Here we test the role of electrostatic interactions in the kinetic stability by analyzing the effects of salt, pH, and point mutations on model discoidal HDL reconstituted from human apolipoprotein C-1 (apoC-1) and dimyristoyl phosphatidylcholine (DMPC). Circular dichroism, Trp fluorescence, and light scattering data show that molar concentrations of NaCl or Na(2)SO(4) increase the apparent melting temperature of apoC-1:DMPC complexes by up to 20 degrees C and decelerate protein unfolding. Arrhenius analysis shows that 1 M NaCl stabilizes the disks by deltaDeltaG* approximately equal 3.5 kcal/mol at 37 degrees C and increases the activation energy of their denaturation and fusion by deltaE(a) approximately equal deltaDeltaH* approximately equal 13 kcal/mol, indicating that the salt-induced stabilization is enthalpy-driven. Denaturation studies in various solvent conditions (pH 5.7-8.2, 0-40% sucrose, 0-2 M trimethylamine N-oxide) suggest that the salt-induced disk stabilization results from ionic screening of unfavorable short-range Coulombic interactions. Thus, the dominant electrostatic interactions in apoC-1:DMPC disks are destabilizing. Comparison of the salt effects on the protein:lipid complexes of various composition reveals an inverse correlation between the lipoprotein stability and the salt-induced stabilization and suggests that short-range electrostatic interactions significantly contribute to lipoprotein stability: the better-optimized these interactions are, the more stable the complex is.     

Phosphatidylcholine substrate specificity of lecithin: cholestrol acyltransferase-in high density lipoproteins and in lipids dispersions.

Lecithin: cholesterol acyltransferase (LCAT) was more highly activated by apolipoprotein A-I (apoA-I) with dimyristoyl phosphatidylcholine (DMPC) than with dilinoleoyl phosphatidylcholine (DLPC) when lipid dispersion of cholesterol and each phosphatidylcholine was used as a substrate. When the enzyme reactions were activated by whole apolipoproteins of high density lipoproteins (HDL), DLPC was more available to the LCAT reaction than DMPC with high concentrations of apoHDL in an incubation mixture. However, no detectable enzyme reaction was observed with dipalmitoyl phosphatidylcholine (DPPC) under both conditions. On the other hand, all of these phosphatidylcholines acted as substrates of LCAT when they were incorporated into HDL coupled to Sepharose. The order of their relative reactivities to cholesterol was DMPC, DPPC, AND DLPC under the conditions used.     

The refined structure of nascent HDL reveals a key functional domain for particle maturation and dysfunction

The cardioprotective function of high-density lipoprotein (HDL) is largely attributed to its ability to facilitate transport of cholesterol from peripheral tissues to the liver. However, HDL may become dysfunctional through oxidative modification, impairing cellular cholesterol efflux. Here we report a refined molecular model of nascent discoidal HDL, determined using hydrogen-deuterium exchange mass spectrometry. The model reveals two apolipoprotein A1 (apoA1) molecules arranged in an antiparallel double-belt structure, with residues 159-180 of each apoA1 forming a protruding solvent-exposed loop. We further show that this loop, including Tyr166, a preferred target for site-specific oxidative modification within atheroma, directly interacts with and activates lecithin cholesterol acyl transferase. These studies identify previously uncharacterized structural features of apoA1 in discoidal HDL that are crucial for particle maturation, and elucidate a structural and molecular mechanism for generating a dysfunctional form of HDL in atherosclerosis.     

Mechanisms Responsible for the Compositional Heterogeneity of Nascent High Density Lipoprotein

Apolipoprotein (apo) A-I-containing nascent HDL particles produced by the ATP binding cassette transporter A1 have different sizes and compositions. To understand the molecular basis for this heterogeneity, the HDL particles produced by apoA-I-mediated solubilization of phospholipid (PL)/free (unesterified) cholesterol (FC) bilayer membranes in cell and cell-free systems are compared. Incubation of apoA-I with ATP binding cassette transporter A1-expressing baby hamster kidney cells leads to formation of two populations of FC-containing discoidal nascent HDL particles. The larger 11-nm diameter particles are highly FC-enriched (FC/PL = 1.2/1 mol/mol) relative to the smaller 8 nm particles and the cell plasma membrane (FC/PL = 0.4/1). ApoA-I-mediated spontaneous solubilization of either multilamellar or unilamellar vesicles made of a membrane-PL mixture and FC yields discoidal HDL particles with diameters in the range 9–17 nm and, as found with the cell system, the larger particles are relatively enriched in FC despite the fact that all particles are created by solubilization of a common FC/PL membrane domain. The size-dependent distribution of FC among HDL particles is due to varying amounts of PL being sequestered in a boundary layer by interaction with apoA-I at the disc edge. The presence of a relatively large boundary layer in smaller discoidal HDL promotes preferential distribution of phosphatidylserine to such particles. However, phosphatidylcholine and sphingomyelin which are the primary PL constituents of nascent HDL do not exhibit selective incorporation into HDL discs of different sizes. This understanding of the mechanisms responsible for the heterogeneity in lipid composition of nascent HDL particles may provide a basis for selecting subspecies with preferred cardio-protective properties.     

Membrane cholesterol uptake by recombinant lipoproteins

Recombinant lipoproteins, prepared with apo A-I isolated from human high density lipoprotein (HDL) and various phospholipids (PLs), were compared with respect to their ability to remove cholesterol (Chol) from labelled erythrocyte ghost membranes. It was found that uptake of Chol was essentially complete following an 8 h incubation at 37 degrees C. Quantitation of the amount of cholesterol taken up showed that recombinants prepared from bovine brain sphingomyelin (BBSM) or dipalmitoyl phosphatidylcholine (DPPC) acquired the highest proportion of Chol (80-140 mol/mol protein), whereas shorter chain phospholipids like dimyristoyl phosphatidylcholine (DMPC) acquired little or no membrane Chol. Chemical analysis of the incubation products indicated that this latter result was due to loss of PL, presumably to the membrane, with consequent disruption of the recombinant particle. Results with DPPC:A-I recombinants of differing PL/protein ratios and sizes showed that Chol uptake was fairly constant at 0.70 mol Chol/mol PL. It is concluded that discoidal, phospholipid-rich recombinant lipoproteins can effectively take up substantial amounts of Chol from physiological membranes, provided that the PLs utilized form micellar complexes which are capable of retaining their structural integrity during the incubation with the membranes.     

Influence of the long-chain/short-chain amphiphile ratio on lateral diffusion of PEG-lipid in magnetically aligned lipid bilayers as measured via pulsed-field-gradient NMR

Lateral diffusion measurements of polyethylene glycol(PEG)-lipid incorporated into magnetically aligned lipid bilayers, composed of dimyristoyl phosphatidylcholine (DMPC) plus dihexanoyl phosphatidylcholine (DHPC) plus 1 mol % (relative to DMPC) dimyristoyl phosphatidylethanolamine-n-[methoxy(polyethylene glycol)-2000] (DMPE-PEG 2000), were performed using stimulated-echo pulsed-field-gradient proton (¹H) nuclear magnetic resonance spectroscopy. The DMPE-PEG 2000 (1 mol %, 35°C) lateral diffusion coefficient D varied directly with the mole fraction of DMPC, X_DMPC = q/(1+q) where q = DMPC/DHPC molar ratio, decreasing progressively from D = 1.65 × 10⁻¹¹ m² s⁻¹ at q ≈ 4.7 to D = 0.65 × 10⁻¹¹ m² s⁻¹ at q ≈ 2.5. Possible sources of this dependence, including orientational disorder, obstruction, and PEG-lipid sequestration, were simulated using, respectively, a diffusion-in-a-cone model, percolation theory, and a two-phase PEG distribution model. Orientational disorder alone was not capable of reproducing the observations, but in combination with either obstruction or PEG-lipid two-phase distribution models did so satisfactorily. A combination of all three models yielded the most reasonable fit to the observed dependence of lateral diffusion on q. These same effects would be expected to influence lateral diffusion of any bilayer-associating species in such systems.     

Effect of phosphatidylethanolamine on the properties of phospholipid-apolipoprotein complexes

Plasma high density lipoproteins (HDL) are synthesized in intestinal mucosal cells and hepatocytes and are secreted into the blood. Factors influencing the structure and function of these HDL, such as lipid and protein composition, are poorly understood. It appears, however, that intracellular, discoidal HDL are enriched, relative to plasma HDL, in phosphatidylethanolamine (PE), a phospholipid known to generate unusual, nonbilayer structures of putative physiological significance. Although incubation of dimyristoylphosphatidylcholine (DMPC) with apolipoprotein A-I at the gel-liquid crystalline phase transition temperature results in the spontaneous formation of lipid-protein complexes, the presence of proportionately small amounts of PE prevents the formation of such complexes, suggesting that PE profoundly alters the phase properties of the phospholipid bilayers. However, by using a detergent-mediated method for the formation of PE-rich model nascent HDL from phospholipids and apolipoprotein A-I, lipid-protein complexes containing as much as 75% DLPE could be formed, thus demonstrating that the presence of PE causes a kinetic, rather than a thermodynamic, barrier to spontaneous complex formation. The products contained a DLPE:DMPC molar ratio similar to that of the initial incubation mixture; however, as the mole percentage of DLPE increased, the products became less heterogeneous, the buoyant density of the products increased, and the Stokes diameter of the products decreased. Similar results were obtained when dimyristoylphosphatidylethanolamine (DMPE) and dipalmitoylphosphatidylethanolamine (DPPE) were employed in lieu of DLPE. Electron microscopy of complexes containing DLPE and DMPC at a 1:1 molar ratio showed that these particles possessed a discoidal, bilayer morphology similar to that seen with complexes containing only phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of reconstituted discoidal high-density lipoproteins on lipid mobilization in RAW 264.7 and CHOK1 cells

Reconstituted discoidal high‐density lipoproteins (rHDL) resemble nascent HDL, which are formed at the early reverse cholesterol transport steps, and constitute the initial cholesterol (Chol) acceptors from cell membranes. We have used different sized rHDL containing or not Chol, to test their abilities to promote cholesterol and phospholipid efflux from two different cell lines: Raw 264.7 macrophages and CHOK1 cells. All rHDL and lipid‐free apolipoprotein A‐I (apoA‐I) were found to be bound to CHO and RAW cells. In RAW cells, a positive correlation between cellular binding and Chol removal was found for 78 and 96 Å rHDL. Chol‐free rHDL were more effective than Chol‐containing ones in binding to RAW cells and promoting Chol removal. These results were more evident in the 96 Å rHDL. On the other hand, rHDL binding to CHO cells was relatively independent of disc size and Chol content. In spite of the fact that apoA‐I and rHDL promoted Chol efflux from both cellular lines, only in CHOK1 cells this result was also associated to decrease Chol esterification. Among choline‐containing phospholipids, only phosphatidylcholine (PC) (but not sphingomyelin) was detected to be effuxed from both cellular lines. With the only exception of Chol‐free 96 Å discs, the other rHDL as well as apoA‐I promoted PC efflux from RAW cells. Chol‐containing rHDL were more active than Chol‐free ones of comparable size to promote PC efflux from RAW macrophages. Regarding CHO cells, only apoA‐I and Chol‐free 78 Å rHDL were active enough to remove PC. J. Cell. Biochem. 113: 1208–1216, 2012. © 2011 Wiley Periodicals, Inc.     

Studies of synthetic peptide analogs of the amphipathic helix. Correlation of structure with function

The four peptide analogs of the amphipathic helix whose interactions with dimyristoyl phosphatidylcholine were described in the preceding paper were compared with apolipoproteins (apo) A-I and A-II in ability to displace native apolipoprotein from high density lipoprotein (HDL) and in ability to activate lecithin:cholesterol acyltransferase. The rank order of the ability of the four peptide analogs to displace apo-A-I from intact HDL was 18A-Pro-18A greater than 18A greater than des-Val10-18A greater than reverse-18A, the same order suggested in the preceding paper for relative lipid affinities. Modified HDL from which 40% of the apo-A-I had been displaced by 18A was indistinguishable from unmodified HDL in its ability to act as a lecithin:cholesterol acyltransferase substrate. This suggests that the easily displaced apo-A-I molecules in polydisperse HDL are relatively ineffectual as lecithin:cholesterol acyltransferase activators and/or 18A replaces the lecithin:cholesterol acyltransferase activity lost. The peptide analog 18A-Pro-18A was found to be a powerful activator of lecithin:cholesterol acyltransferase when incubated with unilamellar egg phosphatidylcholine (PC) vesicles, reaching 140% of the activity of apo-A-I at a 1:1.75 peptide-to-egg PC ratio. In another experiment, it was found that discoidal egg PC complexes of 18A-Pro-18A, 18A, and des-Val10-18A, formed by cholate dialysis, had 30-45% of the activity of apo-A-I/egg PC discoidal complexes, also formed by cholate dialysis, at the same peptide/lipid weight ratio. Examination of the structures formed when the 18A-Pro-18A peptide was incubated with unilamellar egg PC vesicles indicated that the ability of 18A-Pro-18A to exceed apo-A-I in lecithin:cholesterol acyltransferase activating ability is due to the spontaneous conversion by 18A-Pro-18A of egg PC vesicles to small protein annulus-bilayer disc structures. Apo-A-I, apo-A-II, nor any of the other three peptide analogs of the amphipathic helix studied were able to convert a significant fraction of egg PC unilamellar vesicles to discoidal structures.     

Pressure perturbation calorimetry of apolipoproteins in solution and in model lipoproteins

High‐density lipoproteins (HDLs) are complexes of lipids and proteins (termed apolipoproteins) that remove cell cholesterol and protect from atherosclerosis. Apolipoproteins contain amphipathic α‐helices that have high content (≥1/3) and distinct distribution of charged and apolar residues, adopt molten globule‐like conformations in solution, and bind to lipid surfaces. We report the first pressure perturbation calorimetry (PPC) study of apolipoproteins. In solution, the main HDL protein, apoA‐I, shows relatively large volume contraction, ΔV_unf = ?0.33%, and an apparent reduction in thermal expansivity upon unfolding, Δα_unf ≤ 0, which has not been observed in other proteins. We propose that these values are dominated by increased charged residue hydration upon α‐helical unfolding, which may result from disruption of multiple salt bridges. At 5°C, apoA‐I shows large thermal expansion coefficient, α(5°) = 15·10^?4 K^?1, that rapidly declines upon heating from 5 to 40°C, α(40°) ? α(5°) = ?4·10^?4 K^?1; apolipoprotein C‐I shows similar values of α(5°) and α(40°). These values are larger than in globular proteins. They indicate dominant effect of charged residue hydration, which may modulate functional apolipoprotein interactions with a broad range of their protein and lipid ligands. The first PPC analysis of a protein–lipid complex is reported, which focuses on the chain melting transition in model HDL containing apoA‐I or apoC‐I, dimyristoyl phosphatidylcholine, and 0–20% cholesterol. The results may provide new insights into volumetric properties of HDL that modulate metabolic lipoprotein remodeling during cholesterol transport. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Interaction between phospholipid bilayer membranes and the polyene antibiotic amphotericin B: Lipid state and cholesterol content dependence

The interaction between amphotericin B and egg yolk phosphatidylcholine, dimyristoyl (DMPC) and dipalmitoyl phosphatidylcholine (DPPC) phospholipid bilayer vesicles has been monitored by the circular dichroism (CD) spectra of amphotericin B at a 1 · 10^?5 M concentration. This method has revealed that amphotericin B may be present in a number of different forms depending on the time elapsed after the mixing, the cholesterol content of the vesicles and the vesicles' physical state. Some striking features of these CD detected species are the following: with egg yolk phosphatidylcholine and a molar cholesterol percentage lower than 25, at 25°C several forms are coexistent, their amount is time-dependent; with dipalmitoyl or dimyristoyl phosphatidylcholines without cholesterol or with a cholesterol molar percentage lower than 25, in the gel state, a form different from the former appears very rapidly; with egg yolk phosphatidylcholine, DMPC and DPPC at a molar cholesterol percentage between 25 and 50 a new form is monitored, identical in the three cases and observed in the liquid crystalline state as well as in the gel state. In the case of the three phospholipids without cholesterol a definite interaction with the antibiotic is observed but with different characteristics according to the nature of lipid.With amphotericin B ‘Fungizone’ the same species are monitored but their appearance is much slower.Two explanations are proposed for the origin of the discrepancies between CD and electronic absorption.     

Features of the structure of apolipoprotein A1 as an HDL and complexes with dimyristoylphosphatidylcholine. A method for low temperature ESR-spectroscopy]

Apolipoprotein A1 (apoA1) was labelled with the excess of 2,2,6,6-tetramethylpiperidinyl-N-oxyl-4-(2,4-dichloro-1,3,5- triazinyl-6)-amine at pH 9.8. The products, containing 7.2 (+/- 1) paramagnetic labels per 1 molecule of apoA1, showed spin-spin and dipole-dipole exchange interactions. The ESR spectra of the spin labelled A1 (at 77K) had d1/d values 0.76 (at pH 7.4), 0.59 (in 3 M NaCl), 0.55 (in 3 M guanidinium hydrochloride), 0.44 (in 40% 2-chloroethanol). Micellar complexes of spin labelled apoA1/DMPC (1 : 20 mol/mol and 1 : 190 mol/mol) and HDL containing spin labelled apoA1 were prepared. Comparison of ESR spectra at 77 K showed that apoA1 structure varies in the complexes with different stoichiometry and in spin labelled HDL. These data show the importance of hydrophobic protein-protein interactions for the structure of HDL and synthetic complexes of apoA1 with phosphatidyl choline.     

Apolipoprotein AI tertiary structures determine stability and phospholipid‐binding activity of discoidal high‐density lipoprotein particles of different sizes

Human high‐density lipoprotein (HDL) plays a key role in the reverse cholesterol transport pathway that delivers excess cholesterol back to the liver for clearance. In vivo, HDL particles vary in size, shape and biological function. The discoidal HDL is a 140–240 kDa, disk‐shaped intermediate of mature HDL. During mature spherical HDL formation, discoidal HDLs play a key role in loading cholesterol ester onto the HDL particles by activating the enzyme, lecithin:cholesterol acyltransferase (LCAT). One of the major problems for high‐resolution structural studies of discoidal HDL is the difficulty in obtaining pure and, foremost, homogenous sample. We demonstrate here that the commonly used cholate dialysis method for discoidal HDL preparation usually contains 5–10% lipid‐poor apoAI that significantly interferes with the high‐resolution structural analysis of discoidal HDL using biophysical methods. Using an ultracentrifugation method, we quickly removed lipid‐poor apoAI. We also purified discoidal reconstituted HDL (rHDL) into two pure discoidal HDL species of different sizes that are amendable for high‐resolution structural studies. A small rHDL has a diameter of 7.6 nm, and a large rHDL has a diameter of 9.8 nm. We show that these two different sizes of discoidal HDL particles display different stability and phospholipid‐binding activity. Interestingly, these property/functional differences are independent from the apoAI α‐helical secondary structure, but are determined by the tertiary structural difference of apoAI on different discoidal rHDL particles, as evidenced by two‐dimensional NMR and negative stain electron microscopy data. Our result further provides the first high‐resolution NMR data, demonstrating a promise of structural determination of discoidal HDL at atomic resolution using a combination of NMR and other biophysical techniques.     

Lipid-binding studies of human apolipoprotein A-I and its terminally truncated mutants

Apolipoprotein A-I (apoA-I, 243 amino acids) is the major protein of high-density lipoproteins (HDL) that plays an important structural and functional role in lipid transport and metabolism. The central region of apoA-I (residues 60-183) is predicted to contain exclusively amphipathic alpha-helices formed from tandem 22-mer sequence repeats. To analyze the lipid-binding properties of this core domain, four terminally truncated mutants of apoA-I, Delta(1-41), Delta(1-59), Delta(1-41,185-243), and Delta(1-59,185-243), were expressed in baculovirus infected Sf-9 cells. The effects of mutations on the ability of apoA-I to form bilayer disk complexes with dimyristoyl phosphatidylcholine (DMPC) that resemble nascent HDL were analyzed by density gradient ultracentrifugation and electron microscopy (EM). The N-terminal deletion mutants, Delta(1-41) and Delta(1-59), showed altered lipid-binding ability as compared to plasma and wild-type apoA-I, and in the double deletion mutants, Delta(1-41, 185-243) and Delta(1-59, 185-243), the lipid binding was abolished. Thermal unfolding of variant apoA-I/DMPC complexes monitored by circular dichroism (CD) showed hysteresis and a shift in the melting curves by about -12 degrees C upon reduction in the heating rate from 1.0 to 0.067 K/min. This indicates an irreversible kinetically controlled transition with a high activation energy E(a) = 60 +/- 5 kcal/mol. CD and EM studies of the apoA-I/DMPC complexes at different pH demonstrated that changes in the net charge or in the charge distribution on the apoA-I molecule have critical effects on the conformation and lipid-binding ability of the protein.     

Interaction of human plasma high-density lipoprotein HDL2b with discoidal complexes of dimyristoylphosphatidylcholine and apolipoprotein A-I

The interaction of HDL2b, a major subclass (d = 1.063 - 1.100 g/ml) of human plasma high-density lipoproteins, with discoidal complexes composed of dimyristoylphosphatidylcholine (DMPC) and apolipoprotein A-I (weight ratio, DMPC/apolipoprotein A-I (2.1 - 2.5:1); dimensions, 10.0 x 4.4 nm) was investigated. Incubation at 37 degrees C for 4.5 h of HDL2b with discoidal complexes resulted in a transfer of DMPC from the discoidal complexes to the HDL2b, a release of lipid-free apolipoprotein A-I from the discoidal complexes during such transfer, and a dissociation of some apolipoprotein A-I from the HDL2b surface. The number of discoidal complexes degraded during interaction with HDL2b depended on the initial molar ratio of HDL2b to discoidal complexes. Approximately one molecule of HDL2b was required for the degradation of one discoidal complex particle, and the degradation process appeared limited by the capacity of the HDL2b for uptake of DMPC. Degradation of discoidal complexes was also observed when human plasma LDL (d = 1.006-1.063 g/ml) was substituted for HDL2b in the interaction mixture.