Computational and experimental characterizations of silver nanoparticle-apolipoprotein biocorona.

With the advancement of nanotoxicology and nanomedicine, it has been realized that nanoparticles (NPs) interact readily with biomolecular species and other chemical and organic matter to result in biocorona formation. The field of the environmental health and safety of nanotechnology, or NanoEHS, is currently lacking significant molecular-resolution data, and we set out to characterize biocorona formation through electron microscopy imaging and circular dichroism spectroscopy that inspired a novel approach for molecular dynamics (MD) simulations of protein-NP interactions. In our present study, we developed a novel GPU-optimized coarse-grained MD simulation methodology for the study of biocorona formation, a first in the field. Specifically, we performed MD simulations of a spherical, negatively charged citrate-covered silver nanoparticle (AgNP) interacting with 15 apolipoproteins. At low ion concentrations, we observed the formation of an AgNP-apolipoprotein biocorona. Consistent with the circular dichroism (CD) spectra, we observed a decrease in α-helices coupled with an increase in ß-sheets in apolipoprotein upon biocorona formation.

Mecanismos básicos: estructura, función y metabolismo de las lipoproteínas plasm / No disponible

El objetivo de este capítulo es presentar información básica sobre la fisiología de las lipoproteínas. La fracción proteica de las lipoproteínas consta de varias apolipoproteínas y enzimas cuyas funciones son el transporte y la metabolización de los lípidos. La clasificación de las lipoproteínas se basa en su densidad y se pueden aislar por ultracentrifugación quilomicrones, VLDL, IDL, LDL y HDL. Los quilomicrones y las VLDL transportan los triglicéridos desde el intestino e hígado, respectivamente, hasta los tejidos periféricos. La metabolización de las VLDL origina las IDL y LDL. Las LDL transportan la mayoría del colesterol plasmático a los tejidos extrahepáticos. La HDL moviliza el colesterol de los tejidos periféricos hacia el hígado, donde se elimina en forma de colesterol libre o sales biliares, proceso conocido como transporte reverso de colesterol. El metabolismo de las lipoproteínas puede ser regulado por receptores nucleares que regulan la expresión de genes del metabolismo de triglicéridos y de las apolipoproteínas (AU)

The aim of this work is to present basic information on the lipoprotein physiology. The protein fraction of lipoproteins consists of several apolipoproteins and enzymes whose functions are lipid transport and metabolism. Classification of lipoproteins is based on their density. Chylomicrons, VLDL, IDL, LDL and HDL can be isolated by ultracentrifugation. Both chylomicrons- and VLDL-triglycerides are transported from the intestine and liver, respectively, to the peripheral tissues. The metabolism of VLDL originates IDL and LDL. LDL is the main transporter of cholesterol to extrahepatic tissues. HDL mobilizes cholesterol from peripheral tissues to the liver where it is secreted to bile as free cholesterol or bile salts, a process termed reverse cholesterol transport. Lipoprotein metabolism can be regulated by nuclear receptors that regulate the expression of genes involved in triglyceride and apolipoprotein metabolism (AU)

Whole egg consumption improves lipoprotein profiles and insulin sensitivity to a greater extent than yolk-free egg substitute in individuals with metabolic syndrome.

OBJECTIVE: We investigated if daily egg feeding, along with carbohydrate restriction, would alter lipoprotein metabolism and influence atherogenic lipoprotein profiles and insulin resistance in men and women with metabolic syndrome (MetS). METHODS: In a randomized, single-blind, parallel design, participants consumed either 3 whole eggs/day (EGG, n=20) or the equivalent amount of yolk-free egg substitute (SUB, n=17), as part of a moderately carbohydrate-restricted diet (25%-30% energy) for 12 weeks. Plasma lipids, apolipoproteins (apos), oxidized LDL (oxLDL), cholesteryl ester transfer protein (CETP) and lecithin-cholesterol acyltransferase (LCAT) activities were assessed at baseline and week 12. Lipoprotein particle concentrations and sizes were measured by nuclear magnetic resonance spectroscopy. RESULTS: Atherogenic dyslipidemia improved for all individuals as evidenced by reductions in plasma triglycerides, apoC-III, apoE, oxLDL, VLDL particle diameter, large VDL, total IDL, small LDL, and medium LDL particles (P<0.05). Furthermore, there were increases in HDL-cholesterol, large LDL and large HDL particles (P<0.05) for all individuals. However, there were greater increases in HDL-cholesterol and large HDL particles, and reductions in total VLDL and medium VLDL particles for those consuming EGG compared to SUB (P<0.05). Plasma insulin and insulin resistance (HOMA-IR) were reduced, while LCAT activity, and both HDL and LDL diameters increased over time in the EGG group only (P<0.05). CONCLUSIONS: Incorporating daily whole egg intake into a moderately carbohydrate-restricted diet provides further improvements in the atherogenic lipoprotein profile and in insulin resistance in individuals with MetS.

Atomic force microscopy of ex vivo amyloid fibrils.

Here, we report a study of ex vivo amyloid fibrils formed, respectively, by the Leu174Ser Apolipoprotein A-I (ApoA-I-LS) variant and by ß2-microglobulin (ß2-m) (Relini et al., J. Biol. Chem. 281:16521-16529, 2006; Relini et al., Biochim. Biophys. Acta 1690:33-41, 2004). In the work on ApoA-I-LS, the AFM has been used to characterize and compare the morphologies of amyloid fibrils isolated from two different patients, while in the study on ß2-m our investigation provided important information about the factors that can promote the aggregation in vivo.

[Penetration of lipoproteins and apolipoproteins of very low density into the myocardium and changes of its structure in rat heart perfusion]. / Proniknovenie lipoproteinov i apolipoproteinov ochen' nizkoi plotnosti v miokard i izmeneniia ego struktury pri perfuzii serdtsa krys in vitro.

The method of electron microscopy, involving extra-low density lipoproteins (ELDLP) marked by colloid gold as well as their protein components (i.e. apolipoproteins--apoELDLP), was made use of to show that they are captured by capillary endotheliocytes of the isolated perfusing rat heart. Receptor-mediated endocytosis is the main mechanism of penetration. Within 30 minutes, ELDLP interact with the capillary wall, pass through the endothelial barrier and, from the intersticium, they are captured by tissue microphages, which induces their activity. They have, on the whole, a positive effect on the intactness of muscle cells of the beating heart. During the same time span, apoELDLP remain in endotheliocytes, however, they exert a pronounced negative impact on the myocardium. The capillary endothelium, in whose cells the lysosomal apparatus activates itself and clasmatosis sets on, is affected morphologically most of all. The impairment of the capillary endothelium, development of the perivascular edema and a reduced coronary flow trigger a sequence of events leading to enhanced cytolytic processes in the myocardium due to an activated lysosomal apparatus of cells.

Insect immune activation by apolipophorin III is correlated with the lipid-binding properties of this protein.

Apolipophorin III (apoLp-III) is an exchangeable insect apolipoprotein consisting of five amphipathic alpha-helices. The protein is able to open reversibly on associating with hydrophobic surfaces and plays a role both in lipid transport and induction of immune responses. Point mutations were introduced at positions 66 (N-->D) and/or 68 (K-->E) between helices 2 and 3, a region possibly serving as a hinge for the opening of the molecule when associating with lipids. The lipid-binding properties of the mutant proteins were analyzed and compared with their immune inducing activities. Structural properties of the proteins were studied by far UV circular dichroism spectroscopy and their abilities to form discoidal complexes of dimyristoyl phosphatidylcholine (DMPC) vesicles were investigated. In comparison to wild-type apoLp-III, apoLp-III(N66D/K68E), and apoLp-III(K68E) displayed significantly decreased lipid-binding abilities and immune stimulating activities, while these effects were less noticeable with apoLp-III(N66D). The secondary structure of the double mutant apoLp-III(N66D/K68E) was similar to that of wild-type apoLp-III. A noticeable reduction of alpha-helical content could be observed for the single mutants apoLp-III(N66D) and apoLp-III(K68E), which was accompanied by an increase in percentage amount of beta-turns. The stability of the secondary structure determined by heat denaturation was not affected by mutagenesis. Furthermore, the ability of all proteins to form discoidal complexes of equal size and shape in the presence of dimyristoyl phosphatidylcholine indicated that the mutagenesis did not affect the molecular architecture in the lipid-associated conformation. The relationship between reduced lipid association and reduced immune stimulating activity supports the hypothesis that apoLp-III-induced immune activation is triggered by the conformational change of the protein.

An N-terminal three-helix fragment of the exchangeable insect apolipoprotein apolipophorin III conserves the lipid binding properties of wild-type protein.

Apolipophorin III (apoLp-III) from the greater wax moth Galleria mellonella is an exchangeable insect apolipoprotein that consists of five amphipathic alpha-helices, sharing high sequence identity with apoLp-III from the sphinx moth Manduca sexta whose structure is available. To define the minimal requirement for apoLp-III structural stability and function, a C-terminal truncated apoLp-III encompassing residues 1-91 of this 163 amino acid protein was designed. Far-UV circular dichroism spectroscopy revealed apoLp-III(1-91) has 50% alpha-helix secondary structure content in buffer (wild-type apoLp-III 86%), increasing to essentially 100% upon interactions with dimyristoylphosphatidylcholine (DMPC). Guanidine hydrochloride denaturation studies revealed similar stability properties for wild-type apoLp-III and apoLp-III(1-91). Resistance to denaturation for both proteins increased substantially upon association with phospholipid. In the absence of lipid, wild-type apoLp-III was monomeric whereas apoLp-III(1-91) partly formed dimers and trimers. Discoidal apoLp-III(1-91)-DMPC complexes were smaller in diameter (13.5 nm) compared to wild-type apoLp-III (17.7 nm), and more molecules of apoLp-III(1-91) associated with the complexes. Lipid interaction revealed that apoLp-III(1-91) binds to modified spherical lipoprotein surfaces and efficiently transforms phospholipid vesicles into discoidal complexes. Thus, the first three helices of G. mellonella apoLp-III contain the basic features required for maintenance of the structural integrity of the entire protein.

Functional and structural properties of lipid-associated apolipoprotein J (clusterin).

Apolipoprotein J (apoJ, clusterin) is a multifunctional protein normally associated with lipids in plasma and cerebrospinal fluid, and secreted as lipoparticles by hepatocytes and astrocytes. To investigate whether the structural and functional properties of apoJ are modulated upon binding to lipids, we prepared apoJ high-density lipoprotein (HDL)-like particles employing either synthetic or plasma HDL-derived lipids. The majority of the resulting lipoparticles contained one molecule of apoJ per particle and exhibited the same alpha2 electrophoretic mobility characteristic of apoJ-containing plasma HDL. Particle size seemed to be dependent on the presence of cholesterol in the lipid mixture and ranged from diameters of 10 nm in the presence of cholesterol to 20 nm in the absence of cholesterol. CD analysis and Fourier-transform infrared spectroscopy revealed similar changes in the apoJ secondary structure induced by its incorporation into lipoparticles, namely a decrease in alpha-helix content and an increase in beta-turn structures. Two functional assays, the binding interaction with Alzheimer's amyloid beta peptides and the inhibitory activity of the complement membrane-attack complex, did not detect any changes in apoJ activity following its lipidation (P>0.05). On the contrary, the binding affinity to the cellular receptor megalin was enhanced significantly (P<0.01) after the association with lipids; the K(d) value decreased from 78.8+/-10.7 nM for the delipidated form to 37. 0+/-7.3 nM for apoJ-HDL. Although it is not known whether the structural changes observed are directly responsible for the higher receptor-binding affinity, the data suggest that the complement inhibition and amyloid beta-binding motifs are located in areas of the molecule different from those involved in the apoJ-megalin interaction.

In vitro amyloid fibril formation by synthetic peptides corresponding to the amino terminus of apoSAA isoforms from amyloid-susceptible and amyloid-resistant mice.

Specific proteins of the apolipoprotein serum amyloid (apoSAA) family that are synthesized in large quantities during the acute, early phase of inflammation can serve as the proteinaceous precursors for amyloid fibrils. To model fibrillogenesis in such inflammatory diseases, we have used electron microscopy and X-ray diffraction to examine the structures formed by synthetic peptides corresponding in sequence to the 11 amino-terminal amino acids of murine apoSAA1, apoSAAcej, and apoSAA2 and to the 15 amino-terminal amino acids of apoSAA2. This region is reported to be the major fibrillogenic determinant of apoSAA isoforms. Both in 1 mM Tris buffer and in 35% acetonitrile, 0.1% trifluoracetic acid (ACN/TFA), all of the peptides formed macromolecular assemblies consisting of twisted, approximately 40- to 60-A-thick ribbons, which varied in width from around 40-70 A (for 11-mer apoSAA2 in Tris) up to 900 A (for the other peptides). X-ray diffraction patterns recorded from lyophilized peptides, vapor-hydrated samples, and solubilized/dried samples showed hydrogen bonding and intersheet reflections typical of a beta-pleated sheet conformation. The coherent lengths measured from the breadths of the X-ray reflections indicated that with hydration the growth of the assemblies in the intersheet stacking direction was comparable to that in the hydrogen-bonding direction, and analysis of oriented samples showed that the beta-strands were oriented perpendicular to both the long axis and the face of the assemblies. These X-ray results are consistent with the ribbon- or plate-like morphology of the individual aggregates and emphasize the polymorphic nature of amyloidogenic peptides. Our findings demonstrate that X-ray diffraction measurements on vapor-hydrated or solubilized/dried versus lyophilized, amyloidogenic peptides are a good indicator of their fibrillogenic potential. For example, from the highest to the lowest potential, the peptides examined here were ranked as: Abeta1-28 > Abeta1-40 > apoSAA1 approximately apoSAAcej > apoSAA2 > Abeta17-42. Experiments in which the three different 11-mer apoSAA isoforms were solubilized in ACN/TFA and then combined as binary mixtures showed that the ribbon morphology was not affected but that the extent of hydrogen bonding in the assemblies was substantially reduced. Our observations on the in vitro assembly of apoSAA analogs emphasize that amyloid fibril formation and morphology depend on primary sequence, length of polypeptide chain, the presence of additional fibrillogenic polypeptides, and solvent conditions.

Role of diacylglycerol and apolipophorin-III in regulation of physiochemical properties of the lipophorin surface: metabolic implications.

Manduca Sexta adults insects have two defined lipophorin species of densities 1.09 g/mL, [high-density lipophorin (HDLp)] and 1.02 g/mL [low-density lipophorin (LDLp)], respectively, and a continuous broad range of lipophorin particles of intermediate size and density, intermediate-density lipophorin (IDLp). The transformation of HDLp into IDLp and LDLp is the result of the progressive loading of HDLp with diacylglycerol (DG) and an exchangeable apolipoprotein, apolipophorin-III (apoLp-III). In this paper, we describe the physiochemical changes which occur in the lipophorin surface as a result of the transformation of HDLp into LDLp. (1) The increase in apoLp-III content, from 0 to 16 molecules per particle, is accompanied by a gradual increase in the zeta-potential which, at pH 8.6 ranges from /1.02 mV for lipophorins without apoLp-III to -7.76 mV for lipophorins containing 16 molecules of apoLp-III. (2) As judged by the changes in the partition constant for trimethylammonium diphenylhexatriene and oleic acid, an average 2-fold increase in the size of the lipophorin lipid surface takes place when HDLp is loaded with Dg and transformed into LDLp. (3) These data, as well as the results obtained by end point lipolysis with a triacylglycerol (TG) lipase, indicated that the accessible DG content increases 4-7 times when HDLp is converted in LDLp. (4) Fluorescence polarization of the cationic and anionic lipid probes, trimethylammonium diphenylhexatriene and cis-parinaric acid, embedded in eight different subspecies of lipophorin, containing from 12 to 50% DG, showed a small decrease in the surface lipid order when going from HDLp (25% DG) to LDLp (50% DG). (5) Porcine pancreatic phospholipase A2 was used as a probe of the lipoprotein surface. As the DG content of the lipoprotein increased, a higher enzyme activity against the lipoprotein-phospholipids was observed, with a maximum activity 5-fold higher against LDLp than against HDLp. Overall, the changes observed as the lipoprotein particles are loaded with DG and apoLp-III provide a link between the structure and properties of the lipophorin surface and the physiological roles of HDLp and LDLp particles.

Effect of phospholipase C and apolipophorin III on the structure and stability of lipophorin subspecies.

Four distinct subspecies of the insect hemolymph lipoprotein, lipophorin, that range in diacylglycerol (DAG) content from approximately 100 to 1000 molecules per particle, were treated with phospholipase C. Lipid analysis demonstrated that both phosphatidylcholine and phosphatidylethanolamine were hydrolyzed to DAG. Phospholipase C was used to remove 74-82% of the phospholipid of different lipophorins and these were analyzed for aggregation. Low density lipophorin (LDLp), the largest subspecies, with a diameter of approximately 23 nm, developed turbidity (monitored by sample absorbance at 340 nm) suggesting the formation of lipoprotein aggregates. High density lipophorin-adult (HDLp-A) and high density lipophorin-wanderer 1 (HDLp-W1) also displayed an increase in A340 when incubated with phospholipase C, although the maximal increase observed was considerably less than that for LDLp on a per particle basis. Phospholipase C caused only a minimal increase in A340 in a fourth subspecies, high density lipophorin-wanderer 2 (HDLp-W2), which contains an even lower amount of DAG. Electron microscopy was used to evaluate changes in particle morphology as a result of phospholipid depletion. HDLp-W2 and HDLp-W1 showed signs of progressive aggregation and particle fusion. A similar aggregation/fusion was seen in the case of high density lipophorin adult (HDLp-A) while LDLp samples contained multiple aggregation/fusion foci and resultant very large particles. In the presence of exogenous apolipophorin III (apoLp-III), phospholipase C-induced lipophorin aggregation/fusion was prevented. Electron microscopy of LDLp and HDLp-A samples revealed that apoLp-III-stabilized, phospholipase C-treated particles had a morphology similar to that of control particles.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron-microscopic and hydrodynamic characterization of recombinant apolipoprotein (a) and its association with LDL.

A recombinant apo(a) containing 17 kringle 4 domains as well as the kringle 5 and protease domains of apo(a) was characterized by hydrodynamic studies and electron microscopy. Recombinant apo(a) is a monomer in solution with a molecular weight of 325,000 by sedimentation equilibrium and 320,000 by sedimentation and diffusion, and it is a highly asymmetric molecule with a frictional ratio of 2.2. In the electron microscope recombinant apo(a) is visualized as a flexible chain of domains approximately 800 A long. Sedimentation velocity studies also demonstrate that when it is mixed with LDL, recombinant apo(a) reversibly forms an Lp(a)-like complex with a 1:1 stoichiometry; moreover, complex formation is inhibited by 6-amino hexanoic acid. Hydrodynamic modeling and electron microscopy suggest that only a small portion of the r-apo(a) molecule interacts with the LDL and the rest of the chain extends into solution. Preliminary studies indicate that recombinant apo(a) also binds mouse LDL.

Synthetic model peptides for apolipoproteins. II. Characterization of the discoidal complexes generated between phospholipids and synthetic model peptides for apolipoproteins.

The structure, composition and physico-chemical properties of complexes generated between phospholipids and synthetic model peptides for the amphipathic helices of the plasma apolipoproteins were studied. The sequences of the peptides were derived from that of the 18A peptide and designed to either enhance or decrease ionic interactions between pairs of peptides, as described in the accompanying paper. Complexes were prepared with dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), or with DPPC and cholesterol, and isolated on a Superose 6HR column. Association kinetics for the DMPC-peptides complexes were followed by measuring the turbidity as a function of the temperature. The diameters of the DPPC-peptide complexes, measured by gradient gel electrophoresis (GGE), were about 120 A. Fluorescence polarization measurements after labeling with diphenyl hexatriene (DPH) yielded transition temperatures of, respectively, 40.6, 41.5 and 41.8 degrees C for the DPPC/18AM1-, DPPC/18AM4- and DPPC/18A-peptide complexes. These values were confirmed by differential scanning calorimetry. Circular dichroism and infrared spectroscopy revealed that the peptides adopt an alpha-helical structure in solution and this percentage increased from 30-40% in the free peptides up to 50-60% in the complexes. Attenuated total reflection (ATR) infrared measurements of the complexes indicated that the peptides are oriented parallel to the acyl chains of the phospholipid bilayer. Denaturation of the peptides and of the peptide-lipid complexes was monitored by Trp fluorescence under addition of increasing amounts of GdmCl. The mid-points of the denaturation curves lie at, respectively, 0.05, 0.25 and 0.35 M GdmCl for the 18AM4, 18A and 18AM1 peptide and are shifted towards higher GdmCl concentrations after peptide-lipid binding. GdmCl denaturation decreased the alpha-helical content of the peptides and of the complexes, as monitored by circular dichroism measurement. The helix to random coil structure transition occurred at, respectively, 2.1, 2.2, and 2.0 M GdmCl for 18A, 18AM1 and 18AM4, compared to 5.1, 5.0, and 5.3 M in the corresponding complexes. These data suggest altogether that the structural properties, the mode of lipid-protein association and the stability of the phospholipid-peptide complexes are similar to those of native plasma apolipoproteins. The 18A and 18AM4 peptides which contain charged residues along the edge of the helix, leading to salt bridge formation between peptides were shown to mimic the amphipathic helices of the plasma apolipoproteins.

Effects of surfactant apolipoproteins on liposome structure: implications for tubular myelin formation.

Tubular myelin is one of several forms of lung surfactant and may play an important role in its surface activity. To determine possible mechanisms of tubular myelin formation, we studied the effects of purified surfactant proteins (SP-A, SP-B, and SP-C) on large unilamellar dipalmitoylphosphatidylcholine-egg phosphatidylglycerol (7/3; wt/wt) liposomes. We studied different types of membrane interaction induced by the apolipoproteins and correlated these with the observed changes in ultrastructure. Aggregation was assessed by measurement of light absorbance, lysis, and fusion by measurement of the fluorescence emitted by water-soluble and lipid-soluble probes, respectively. Mixtures of the apolipoproteins and liposomes were examined in ultrastructural studies by negative staining and by thin sectioning. We found that each protein had a pronounced and distinct effect on liposome structure. SP-A caused aggregation, whereas SP-B and SP-C also caused extensive leakage of liposome contents (lysis) and some degree of lipid mixing (fusion). The disruptive effects of SP-B and to a lesser extent those of SP-C were correlated by negative staining with the appearance of bilayer disks, which tended to aggregate into large sheets. There was a marked synergy between SP-A and SP-B in the process of membrane fusion in the presence of calcium, which correlated with an early (10 min) and extensive rearrangement of the structures seen by electron microscopy followed by a delayed (24 h) appearance of small amounts of tubular myelin.

[Genes of the lipase family: comparison of nucleic and proteinic sequences]. / Les gènes de la famille des lipases: comparaison des séquences nucléiques et protéiques.

Vertebrates' plasmatic apolipoproteins and a few number of lipases in their metabolism present sequence homologies. They are grouped in genes families. The four exons apolipoproteins gene family includes nine human genes: the divergence rate of their sequences allows to place the first ancestral gene very high in the phylogenetic tree of the evolution. However, a more recent duplication of apolipoprotein C-I gene dating from 40 millions years, may be a phylogenetic marker for the radiation of Monkeys. Pancreatic lipase and isoforms, lipoprotein-lipase and hepatic triacylglycerol-lipase form by their homologies a "superfamily" of genes, which also includes yolk proteins of Dipterians eggs. Sequence homologies of PL, LPL and HL are analysed and compared with multiple alignments of amino-acids and nucleotides on spreadsheets. From these comparisons we may characterize four classes of phylogenetic markers: 1) repetitive DNA sequence (Alu, B1, PRE-1) appeared during Mammals evolution, 2) short insertions or deletions (within N-terminal domain) and a gene conversion in guinea-pig lineage, 3) a progressive reduction of intron number during the lipases evolution, 4) several duplications of genes which have produced the five genes of this superfamily currently known in the human genome.

Nuclear magnetic resonance investigation of the interactions with phospholipid of an amphipathic alpha-helix-forming peptide of the apolipoprotein class.

To further understand the packing of amphipathic alpha-helices of apolipoproteins in serum lipoproteins, we have investigated the interactions with dimyristoyl phosphatidylcholine (DMPC) of a 13C-labeled, 18-residue peptide (18A) which can form an amphipathic alpha-helix. This peptide whose amino acid sequence is DWLKAFYDKVAEKLKEAF has the positive-negative residue clustering typical of the apolipoprotein class of amphipathic helix. 13CH3-alanine was introduced as the 11th residue of 18A so that the 13CH3 group protrudes on the apolar side of the amphipathic helix. [13C]NMR spectra of [13C-Ala11]18A in discoidal complexes with DMPC show three resonances from the Ala-13CH3 group; one originates from 18A in aqueous solution, while those at chemical shifts (delta) of 15.2 and 16.4 ppm are assigned to 18A in the "edge" and "faces," respectively, of the discoidal complex. The proportion of 18A in the faces of the discoidal complex increases as the size of the disk is increased by raising the lipid/peptide ratio. 18A covers the edge of the disk so that the 13CH3-Ala side chain from these molecules is in contact with DMPC acyl chains. [13C-Ala11]18A bound to the surface of an egg PC small unilamellar vesicle gives a single resonance from 18A at delta 16.3 ppm consistent with there being no edge location. Cooling 18A-DMPC disks to 15 degrees C crystallizes the DMPC bilayer and restricts the motion of the 13CH3-Ala group of the 18A molecules. The molecular motions of the side chains of the amphipathic helix are sensitive to their location in the disk and to PC molecular packing.

Mode of assembly of amphipathic helical segments in model high-density lipoproteins.

The structure of discoidal apo A-I-phospholipid complexes, representing the metabolic precursors of mature high-density lipoprotein particles, was studied by a combination of both a theoretical and an experimental approach. The secondary structure of the complex was determined by circular dichroic measurements, while the relative orientation of the apo A-I helical segments and of the phospholipid acyl chains was determined by ATR infrared measurements. Fluorescence energy transfer between the tryptophan residues of apo A-I and fluorescent phospholipid probes yielded an estimation of the relative topography of the lipid and apolipoprotein components in discoidal and spherical particles. The theoretical approach consisted of the identification of the helical segments in various apo A-I species. These segments were then oriented at a lipid/water interface by minimization of their hydrophobic and hydrophilic transfer energies. The calculation of the hydrophobicity profiles along the axis of the helices leads to the identification of specific interactions between pairs of helices. The helices were further assembled together with the phospholipids by computer modelling, enabling an estimation of the dimensions of the complex. The combination of the experimental and theoretical results yielded a model for discoidal apolipoprotein-phospholipid complexes, in which the amphipathic helical segments are oriented along the edges of the discs. Such a model can be extended to the conversion of these complexes into mature spherical HDL, through the formation of a cholesteryl ester core.