Apolipoprotein E: phospholipid binding studies with synthetic peptides from the carboxyl terminus.

We have previously shown that the synthetic peptide apoE(129-169) forms lipid-peptide complexes with dimyristoylphosphatidylcholine (DMPC) with an L:P molar ratio of 125:1; the peptide in the isolated complex contains approximately 56% alpha-helicity. These results verify the presence of an amphipathic alpha-helix in this region of apoE as predicted by Chou-Fasman analysis and hydrophobicity calculations. To further define the lipid binding regions of apoE, we have synthesized four peptides, apoE(211-243), -(202-243), -(267-286), and -(263-286), from the carboxyl terminus of apoE and studied their lipid binding properties; apoE(202-243) contains two potential amphipathic helices. Although all four peptides formed alpha-helices in the helix-forming solvent 30% hexafluoropropanol, we found that only apoE(263-286) formed a stable complex with DMPC. The peptide contained approximately 80% alpha-helicity, and its Trp fluorescence spectrum was blue-shifted by 20 nm in the complex which had an L:P ratio of 163:1. We conclude that this sequence is a newly identified lipid binding region of apoE and that the amphipathic helices 203-221 and 226-243 are too hydrophilic to bind phospholipid.     

Motion and surface accessibility of spin-labeled lipids in a model lipoprotein containing cholesteryl oleate, dimyristoylphosphatidylcholine, and apolipoprotein E

A series of spin-labeled phosphatidylcholines (PCs) and cholesteryl esters (CEs) bearing the paramagnetic 2,2-dimethyloxazolidinyl-1-oxy (doxyl) group at fatty acyl carbon C5', C12', or C16' were used to study acyl chain motions in the polar surface shell and hydrophobic core domains of microemulsion (ME) particles containing cholesteryl oleate and dimyristoylphosphatidylcholine (DMPC), and of particles with apolipoprotein E (apoE) bound to their surfaces. Electron paramagnetic resonance data obtained with the doxyl-labeled PCs indicated a gradient of motion in the ME surface monolayer similar to that observed with the same probes in a bilayer. The 5- and 12-doxyl-CEs clearly demonstrated a higher degree of order for the cholesteryl ester rich core than the corresponding doxyl-PCs showed for the phospholipid-rich surface over the entire range 10-60 degrees C. The temperature dependencies of spectra of the 16-doxyl-CE in the core and PC in the surface of the ME were almost identical, suggesting that there was no sharp boundary between core and surface domains. None of the probes detected either the surface phospholipid transition (31 degrees C) or the cholesteryl ester core transition (46 degrees C) measured previously by differential scanning calorimetry and 13C nuclear magnetic resonance. Binding of apoE to spin-labeled DMPC vesicles increased the order of the 5'-position of the sn-2 acyl chain over the range 15-33 degrees C; the thermal transition was broadened and its midpoint elevated. The effect of protein binding was not as striking for the ME particles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anionic phospholipids inhibit apolipoprotein E--low-density lipoprotein receptor interactions

Apolipoprotein E (apoE) is a ligand for members of the low-density lipoprotein receptor (LDLR) family. Lipid-free apoE is not recognized by LDLR, yet interaction with lipid confers receptor recognition properties. Although lipid interaction is known to induce a conformational change in apoE, it is not known if the lipid composition of the resulting complex influences binding. Using reconstituted lipoprotein particles of apoE3 N-terminal (NT) domain and dimyristoylphosphatidylcholine (DMPC), maximal LDLR binding was observed at DMPC:apoE3-NT ratios >2.5:1 (w/w). ApoE3-NT lipid particles prepared with egg sphingomyelin were functional as LDLR ligands while complexes formed with the anionic phospholipids dimyristoylphosphatidylglycerol or dimyristoylphosphatidylserine (DMPS) were not. In the case of apoE3-NT, lipid particles comprised of a mixture of DMPC and DMPS, a DMPS concentration dependent inhibition of LDLR binding activity was observed. Thus, in addition to affecting apoE conformational status, the lipid composition of ligand particles can modulate LDLR binding activity.     

Microemulsions of cholesteryl oleate and dimyristoylphosphatidylcholine: a model for cholesteryl ester rich very low density lipoproteins

This study describes the preparation, purification, and characterization of a cholesteryl oleate/dimyristoylphosphatidylcholine microemulsion as a model for the interaction of lipid domains in cholesteryl ester rich very low density lipoproteins. These lipids were chosen specifically because their thermal transitions were distinct from each other, and their differences in chemical structure permitted the motion(s) of each lipid component to be monitored independently by 13C nuclear magnetic resonance (NMR). The model particles were formed by cosonication of cholesteryl oleate and dimyristoylphosphatidylcholine in a 4:1 molar ratio for 45 min at 55-60 degrees C (above both lipid phase transition temperatures). The crude microemulsion was fractionated by low-speed centrifugation and Sepharose CL-2B chromatography. Microemulsion particles which eluted from the column at a volume similar to that of cholesteryl ester rich very low density lipoproteins had high cholesteryl ester:phospholipid ratios (2.5:1----6:1). Electron micrographs of negatively stained particles showed them to be large spheres devoid of multilamellar or unilamellar vesicle structures. Particle size calculated from a simple compositional model correlated well with sizes determined by electron microscopy (500-1000 A) for various column fractions. Differential scanning calorimetry studies of the microemulsion revealed two thermal transitions for the model particles, at 31.0 and 46.6 degrees C, which were tentatively assigned to the surface phospholipid and core cholesteryl ester domains, respectively. These assignments were confirmed by 13C NMR which demonstrated that, at temperatures near the lower thermotropic transition, only resonances derived from carbon atoms of dimyristoylphosphatidylcholine (DMPC) were observable. As the temperature was raised to 38.6 degrees C, resonances from the olefinic carbons in the cholesteryl ester acyl chain appeared in the spectrum. At 46.6 degrees C, the center of the higher temperature endotherm, resonances from both the steroid ring and remaining acyl chain carbons of cholesteryl oleate became observable in the spectrum. Further increases in temperature did not result in the appearance of new resonances; however, those that were present narrowed and increased in intensity. The elevation in transition temperature for DMPC in these particles (31 degrees C) as compared to that for DMPC in small unilamellar (18 degrees C) and large multilamellar (23 degrees C) vesicles suggested a stabilization of the phospholipid monolayer, possibly by interaction with the nonpolar core lipids.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differences in the binding capacity of human apolipoprotein E3 and E4 to size-fractionated lipid emulsions.

We describe sensitive new approaches for detecting and quantitating protein-lipid interactions using analytical ultracentrifugation and continuous size-distribution analysis [Schuck (2000) Biophys. J.78, 1606-1619]. The new methods were developed to investigate the binding of human apolipoprotein E (apoE) isoforms to size-fractionated lipid emulsions, and demonstrate that apoE3 binds preferentially to small lipid emulsions, whereas apoE4 exhibits a preference for large lipid particles. Although the apparent binding affinity for large emulsions is similar (Kd approximately 0.5 micro m), the maximum binding capacity for apoE4 is significantly higher than for apoE3 (3.0 and 1.8 amino acids per phospholipid, respectively). This indicates that apoE4 has a smaller binding footprint at saturation. We propose that apoE isoforms differentiate between lipid surfaces on the basis of size, and that these differences in lipid binding are due to a greater propensity of apoE4 to adopt a more compact closed conformation. Implications for the role of apoE4 in blood lipid transport and disease are discussed.     

Domains of apoE required for binding to apoE receptor 2 and to phospholipids: implications for the functions of apoE in the brain

We have studied the contribution of the carboxy terminal domains of lipid-free apoE isolated from apoE-expressing cell cultures in binding to phospholipids and have determined the affinities of reconstituted POPC-apoE particles for the apoER2. It was found that the initial rate of association of apoE2, apoE3, apoE4, and a mutant form apoE4R158M to multilamellar DMPC vesicles was similar and was reduced and eventually diminished by gradual deletion of the carboxy terminal segments. The truncated apoE forms retained their ability to associate with plasma lipoproteins. Receptor binding studies were performed using the ldlA-7 cells expressing apoER2 and transiently transfected COS-M6 and the appropriate control untransfected cells. Specific binding to apoER2 was obtained by subtracting from the total binding to the receptor-expressing cells the nonspecific binding values of the untransfected cells. POPC-apoE particles generated using apoE3, apoE4, the truncated apoE4-259, apoE4-229, apoE4-202, and apoE-165, and the mutant apoE4R158M all bound tightly to the apoER2 (K(d) range of 12 +/- 3 to 19 +/- 4 microg/mL). POPC-apoE2 bound with reduced affinity (K(d) = 31 +/- 5.3 microg/mL). The findings establish that the apoER2 binding domain of apoE is in the 1-165 amino terminal region, whereas the carboxy terminal 230-299 region of apoE is required for efficient initial association with phospholipids.     

Conformational reorganization of the four-helix bundle of human apolipoprotein E in binding to phospholipid

Conformational reorganization of the amino-terminal four-helix bundle (22-kDa fragment) of apolipoprotein E (apoE) in binding to the phospholipid dimyristoylphosphatidylcholine (DMPC) to form discoidal particles was investigated by introducing single, double, and triple interhelical disulfide bonds to restrict the opening of the bundle. Interaction of apoE with DMPC was assessed by vesicle disruption, turbidimetric clearing, and gel filtration assays. The results indicate that the formation of apoE.DMPC discoidal particles occurs in a series of steps. A triple disulfide mutant, in which all four helices were tethered, did not form complexes but could release encapsulated 5-(6)-carboxylfluorescein from DMPC vesicles, indicating that the initial interaction does not involve major reorganization of the helical bundle. Initial interaction is followed by the opening of the four-helix bundle to expose the hydrophobic faces of the amphipathic helices. In this step, helices 1 and 2 and helices 3 and 4 preferentially remain paired, since these disulfide-linked mutants bound to DMPC in a manner similar to that of the 22-kDa fragment of apoE4. In contrast, mutants in which helices 2 and 3 and/or helices 1 and 4 paired bound poorly to DMPC. However, all single and double helical pairings resulted in the formation of larger discs than were formed by the 22-kDa fragment, indicating that further reorganization of the helices occurs following the initial opening of the four-helix bundle in which the protein assumes its final lipid-bound conformation. In support of this rearrangement, reducing the disulfide bonds converted the large disulfide mutant discs to normal size.     

Structural determinants in the C-terminal domain of apolipoprotein E mediating binding to the protein core of human aortic biglycan

Apolipoprotein (apo) E-containing high density lipoprotein particles were reported to interact in vitro with the proteoglycan biglycan (Bg), but the direct participation of apoE in this binding was not defined. To this end, we examined the in vitro binding of apoE complexed with dimyristoylphosphatidylcholine (DMPC) to human aortic Bg before and after glycosaminoglycan (GAG) depletion. In a solid-phase assay, apoE.DMPC bound to Bg and GAG-depleted protein core in a similar manner, suggesting a protein-protein mode of interaction. The binding was decreased in the presence of 1 m NaCl and was partially inhibited by either positively (0.2 m lysine, arginine) or negatively charged (0.2 m aspartic, glutamic) amino acids. A recombinant apoE fragment representing the C-terminal 10-kDa domain, complexed with DMPC, bound as efficiently as full-length apoE, whereas the N-terminal 22-kDa domain was inactive. Similar results were obtained with a gel mobility shift assay. Competition studies using a series of recombinant truncated apoEs showed that the charged segment in the C-terminal domain between residues 223 and 230 was involved in the binding. Overall, our results demonstrate that the C-terminal domain contains elements critical for the binding of apoE to the Bg protein core and that this binding is ionic in nature and independent of GAGs.     

Effects of lipid interaction on the lysine microenvironments in apolipoprotein E

Lysines in apolipoprotein (apo) E are key factors in the binding of apoE to the low density lipoprotein receptor, and high affinity binding requires that apoE be associated with lipid. To gain insight into this effect, we examined the microenvironments of the eight lysines in the 22-kDa fragment of apoE3 (residues 1-191) in the lipid-free and lipid-associated states. As shown by (1)H,(13)C heteronuclear single quantum coherence nuclear magnetic resonance, lysine resonances in the lipid-free fragment were poorly resolved over a wide pH range, whereas in apoE3.dimyristoyl phosphatidylcholine (DMPC) discs, the lysine microenvironments and protein conformation were significantly altered. Sequence-specific assignments of the lysine resonances in the spectrum of the lipidated 22-kDa fragment were made. In the lipid-free protein, six lysines could be resolved, and all had pK(a) values above 10. In apoE3.DMPC complexes, however, all eight lysines were resolved, and the pK(a) values were 9.2-11.1. Lys-143 and Lys-146, both in the receptor binding region in helix 4, had unusually low pK(a) values of 9.5 and 9.2, respectively, likely as a result of local increases in positive electrostatic potential with lipid association. Shift reagent experiments with potassium ferricyanide showed that Lys-143 and Lys-146 were much more accessible to the ferricyanide anion in the apoE3.DMPC complex than in the lipid-free state. The angle of the nonpolar face of helix 4 is smaller than the angles of helices 1, 2, and 3, suggesting that helix 4 cannot penetrate as deeply into the DMPC acyl chains at the edge of the complex and that its polar face protrudes from the edge of the disc. This increased exposure and the greater positive electrostatic potential created by interaction with DMPC may explain why lipid association is required for high affinity binding of apoE to the low density lipoprotein receptor.     

Characterization of low density lipoprotein receptor ligand interactions by fluorescence resonance energy transfer

The low density lipoprotein receptor (LDLR) is the prototype of a family of cell surface receptors involved in a wide range of biological processes. A soluble low density lipoprotein receptor (sLDLR) and a tryptophan (Trp)-deficient variant human apolipoprotein E3 (apoE3) N-terminal domain (NT) were used in binding studies. The sole cysteine in apoE3-NT was covalently modified with an extrinsic fluorescence probe, N-(iodoacetyl)-N'-(5-sulfo-1-napthyl)ethylenediamine (AEDANS), and the protein was complexed with lipid. Incubation of sLDLR with AEDANS-Trp-null apoE3-NT dimyristoylphosphatidylcholine (DMPC) disks, but not lipid-free AEDANS-apoE, induced an enhancement in AEDANS fluorescence emission intensity (excitation, 280 nm) consistent with intermolecular energy transfer from excited Trp in sLDLR to receptor-bound apoE. Ligand binding to sLDLR required calcium and was saturable. In competition binding assays, unlabeled apoE3-NT DMPC inhibited AEDANS-apoE DMPC binding to sLDLR more effectively than low density lipoprotein. Fluorescence changes in this system reflected pH-dependent ligand binding and release from sLDLR consistent with models derived from the X-ray crystal structure of the receptor at endosomal pH. Intermolecular energy transfer from excited Trp in LDLR family members to fluorescently tagged ligands represents a sensitive and convenient assay for the characterization of the myriad molecular interactions ascribed to this family of receptor.     

Biophysical properties of apolipoprotein E4 variants: implications in molecular mechanisms of correction of hypertriglyceridemia

In humans and animal models, high plasma concentrations of apolipoprotein (apo) E are associated with hypertriglyceridemia. It has been shown that overexpression of human wild-type (WT) apoE4 in apoE-deficient mice induces hypertriglyceridemia. In contrast, overexpression of an apoE4 variant, apoE4-mut1 (apoE4(L261A, W264A, F265A, L268A, V269A)), does not induce hypertriglyceridemia and corrects hypercholesterolemia. Furthermore, overexpression of another variant, apoE4-mut2 (apoE4(W276A, L279A, V280A, V283A)), induces mild hypertriglyceridemia and does not correct hypercholesterolemia. To better understand how these mutations improve the function of apoE4, we investigated the conformation and stability of apoE4-mut1 and apoE4-mut2 and their binding to dimyristoyl phosphatidylcholine (DMPC) vesicles and to triglyceride (TG)-rich emulsion particles. We found that the mutations introduced in apoE4-mut1 lead to a more stable and compactly folded conformation of apoE4. These structural changes are associated with a slower rate of solubilization of DMPC vesicles by apoE4-mut1 and reduced binding of the protein to emulsion particles compared with WT apoE4. Under conditions of apoE4 overexpression, the reduced binding of apoE4-mut1 to TG-rich lipoprotein particles may facilitate the lipolysis of these particles and may alter the conformation of the lipoprotein-bound apoE in a way that favors the efficient clearance of the lipoprotein remnants. Mutations introduced in apoE4-mut2 result in smaller structural alterations compared with those observed in apoE4-mut1. The slightly altered structural properties of apoE4-mut2 are associated with slightly reduced binding of this protein to TG-rich lipoprotein particles and milder hypertriglyceridemia as compared with WT apoE4.     

Association of apolipoprotein E with the low density lipoprotein receptor: demonstration of its co-operativity on lipid microemulsion particles

When human apolipoprotein E (apoE), which forms a self-associated tetramer in an aqueous solution, bound to the surface of triolein/phosphatidylcholine microemulsion with a particle diameter of 26 nm, it became monomeric on the lipid particle surface without strong evidence for its accumulation on a particular particle that might be expected from its tetramer formation in the aqueous phase. ApoE in the form of the self-associated tetramer did not inhibit binding of human low density lipoprotein (LDL) to its receptor on cultured human skin fibroblast. LDL binding was inhibited only when apoE was bound to the lipid particle surface. The affinity of the apoE-containing lipid particle to the LDL receptor was of the same order as that of LDL on the basis of particle molarity when the surface of the particle was covered with apoE up to 40 to 50% of the saturation level. When the particle was covered more with apoE, the affinity increased by some 20 times. Since the surface of the lipid particle was saturated with 7 apoE molecules, the particle seemed to require to have at least 4 apoE molecules on its surface in order to obtain high binding affinity to LDL receptor.     

Lipid binding ability of human apolipoprotein E N-terminal domain isoforms: correlation with protein stability?

Human apolipoprotein (apo) E exists as one of three major isoforms, E2, E3 or E4. Individuals carrying the 4 allele have an increased risk of heart disease and premature onset of Alzheimer's disease. To investigate the molecular basis for this phenomenon, the N-terminal domain of apoE3, apoE2 and apoE4 were expressed in bacteria, isolated and employed in lipid binding and stability studies. Far UV circular dichroism spectroscopy in buffer at pH 7 revealed a similar amount of -helix secondary structure for the three isoforms. By contrast, differences were noted in apoE-NT isoform-specific transformation of bilayer vesicles of dimyristoylphosphatidylglycerol (DMPG) into discoidal complexes. ApoE4-NT induced transformation was most rapid, followed by apoE3-NT and apoE2-NT. To determine if differences in the rate of apoE-NT induced DMPG vesicle transformation is due to isoform-specific differences in helix bundle stability, guanidine HCl denaturation studies were conducted. The results revealed that apoE2-NT was the most stable, followed by apoE3-NT and apoE4-NT, establishing an inverse correlation between helix bundle stability and DMPG vesicle transformation rate at pH 7. When the zwitterionic dimyristoylphosphatidylcholine (DMPC) was employed as the model lipid surface, interaction of apoE-NT isoforms with the lipid substrate was slow. However, upon lowering the pH from 7 to 3, a dramatic increase in the rate of DMPC vesicle transformation rate was observed for each isoform. To evaluate if the increased DMPC vesicle transformation rates observed at low pH is due to pH-dependent alterations in helix bundle stability, guanidine HCl denaturation studies were performed. ApoE2-NT and apoE3-NT displayed increased resistance to denaturation as a function of decreasing pH, while apoE4-NT showed no change in stability. Studies with the fluorescent probe, 8-anilino-1-naphthalene sulfonic acid, indicated an increase in apoE hydrophobic surface exposure upon decreasing the pH to 3.0. Taken together, the data indicate that changes in the stability of secondary structure elements in apoE-NT isoforms are not responsible for pH-induced increases in lipid binding activity. It is likely that pH-induced disruption of inter-helical tertiary contacts may promote helix bundle conformational changes that present the hydrophobic interior of the protein to potential lipid surface binding sites.     

High affinity binding of human interleukin 4 to cell lines

Purified human recombinant interleukin 4 (IL-4) was radio iodinated to high specific radioactivity without loss of biological activity. 125I-IL-4 bound specifically to the Burkitt lymphoma Jijoye cells and other cell lines. Jijoye cells showed a high affinity for 125I-IL-4 (Kd approximately equal to 7 10(-11) M) and displayed 1200-1400 specific receptors per cell at 4 degrees C or 37 degrees C. The equilibrium dissociation constant (Kd) corresponds to the IL-4 concentration which induces 50% maximal expression of the low affinity IgE receptor (Fc epsilon RL/CD23) on Jijoye cells. At 4 degrees C the rate constant of association K1 is 1.7 x 10(6) M-1 s-1 and the rate contant of dissociation k -1 is 1.3 x 10(-4) s-1 (t 1/2 = 91 min.) No human recombinant lymphokines other than IL-4 were able to compete for the binding of 125I-IL-4 to its receptor.     

Structure-guided protein engineering modulates helix bundle exchangeable apolipoprotein properties

Apolipoprotein (apo) E plays a major role in lipid metabolism by mediating cellular uptake of lipoprotein particles through interaction with members of the low density lipoprotein (LDL) receptor family. The primary region of apoE responsible for receptor binding has been limited to a cluster of basic amino acids between residues 134 and 150, located in the fourth helix of the N-terminal domain globular helix bundle structure. To investigate structural and functional requirements of this "receptor binding region" we engineered an apolipoprotein chimera wherein residues 131-151 of human apoE were substituted for residues 146-166 (helix 5) of Manduca sexta apolipophorin III (apoLp-III). Recombinant hybrid apolipoprotein was expressed in Escherichia coli, isolated, and characterized. Hybrid apolipoprotein and apoE3-N-terminal, but not apoLp-III, bound to heparin-Sepharose. Far UV circular dichroism spectroscopy revealed the presence of predominantly alpha-helix secondary structure, and stability studies revealed a urea denaturation midpoint of 1.05 m, similar to wild-type apoLp-III. Hybrid apolipoprotein-induced dimyristoylphosphatidylcholine (DMPC) bilayer vesicle solubilization activity was significantly enhanced compared with either parent protein, consistent with detection of solvent-exposed hydrophobic regions on the protein in fluorescent dye binding experiments. Unlike wild-type apoLp-III.DMPC complexes, disc particles bearing the hybrid apolipoprotein competed with 125ILDL for binding to the LDL receptor on cultured human skin fibroblasts. We conclude that a hybrid apolipoprotein containing a key receptor recognition element of apoE preserves the structural integrity of the parent protein while conferring a new biological activity, illustrating the potential of helix swapping to introduce desirable biological properties into unrelated or engineered apolipoproteins.     

Apolipoprotein E: phospholipid binding studies with synthetic peptides containing the putative receptor binding region

To define the lipid and receptor binding regions of apolipoprotein E (apoE), we have synthesized four peptides beginning at residue 169 and continuing through the putative receptor binding region and ending at residue 129 so as to include a proposed lipid binding domain. The peptides were synthesized by solid-phase techniques, cleaved with anhydrous HF, and purified by ion-exchange and semipreparative reversed-phase high-performance liquid chromatography (HPLC). The peptides had the correct amino acid composition and were greater than 99% pure by analytical reversed-phase HPLC. The circular dichroic spectrum of each peptide was recorded before and after mixing with dimyristoylphosphatidylcholine. With apoE (148-169), apoE (144-169), and apoE (139-169), no changes were observed in the ellipticity at 222 nm. However, with apoE (129-169), an increase in alpha-helicity to approximately 42% was observed. Density gradient ultracentrifugation of the lipid-peptide mixture permitted isolation of a complex with apoE (129-169) with a molar ratio of lipid to peptide of 125:1, which was stable to recentrifugation. The alpha-helicity of the peptide in the complex was estimated to be 56%. No complexes were isolated from the gradients of the shorter peptides. Therefore, we conclude that the amphipathic helix formed by residues 130-150 contains one of the lipid binding regions of apoE.     

Complex of human apolipoprotein C-1 with phospholipid: thermodynamic or kinetic stability?

Thermal unfolding of discoidal complexes of apolipoprotein (apo) C-1 with dimyristoyl phosphatidylcholine (DMPC) reveals a novel mechanism of lipoprotein stabilization that is based on kinetics rather than thermodynamics. Far-UV CD melting curves recorded at several heating/cooling rates from 0.047 to 1.34 K/min show hysteresis and scan rate dependence characteristic of slow nonequilibrium transitions. At slow heating rates, the apoC-1 unfolding in the complexes starts just above 25 degrees C and has an apparent melting temperature T(m) approximately 48 +/- 1.5 degrees C, close to T(m) = 51 +/- 1.5 degrees C of free protein. Thus, DMPC binding may not substantially increase the low apparent thermodynamic stability of apoC-1, DeltaG(25 degrees C) < 2 kcal/mol. The scan rate dependence of T(m) and Arrhenius analysis of the kinetic data suggest an activation enthalpy E(a) = 25 +/- 5 kcal/mol that provides the major contribution to the free energy barrier for the protein unfolding on the disk, DeltaG > or = 17 kcal/mol. Consequently, apoC-1/DMPC disks are kinetically but not thermodynamically stable. To explore the origins of this kinetic stability, we utilized dynode voltage measured in CD experiments that shows temperature-dependent contribution from UV light scattering of apoC-1/DMPC complexes (d approximately 20 nm). Correlation of CD and dynode voltage melting curves recorded at 222 nm indicates close coupling between protein unfolding and an increase in the complex size and/or lamellar structure, suggesting that the enthalpic barrier arises from transient disruption of lipid packing interactions upon disk-to-vesicle fusion. We hypothesize that a kinetic mechanism may provide a general strategy for lipoprotein stabilization that facilitates complex stability and compositional variability in the absence of high packing specificity.     

Metal binding and metal-induced conformational change of frog bone gamma-carboxyglutamic acid-containing protein

Ca2+ or Cd2+ binding and the conformational change induced by the metal binding in two frog bone Gla-proteins (BGP, termed BGP-1 and BGP-2) were studied by equilibrium dialysis and CD measurement. By CD measurement in the far-ultraviolet region, the alpha-helix content of both apoBGPs was found to be 8%. Binding of both Ca2+ and Cd2+ was accompanied with a change in the CD spectrum, and the alpha-helix content increased to 15 and 25% for BGP-1 and BGP-2, respectively. CD measurement in the near-ultraviolet region indicated that the environment of aromatic amino acid residues in the protein molecule was changed by metal binding. Equilibrium dialysis experiments indicated that each of these two protein binds specifically 2 mol of Ca2+, and nonspecifically an additional 3-4 mol of Ca2+ in 0.02 M Tris-HCl/0.15 M NaCl (pH 7.4), at 4 degrees C. According to the two separate binding sites model, BGP-1 has 1 high-affinity Ca2+ binding site (Kd1 = 0.17 mM) and 1 low-affinity site (Kd2 = 0.29 mM), and BGP-2 contains 1 high-affinity site (Kd1 = 0.14 mM) and 1 low-affinity site (Kd2 = 0.67 mM). In addition, 2 Cd2+ bound to a high-affinity binding site on BGP-1 with Kd1 of 10.4 microM, and 1 Cd2+ bound to a low-affinity binding site with Kd2 of 41.5 microM. On the other hand, BGP-2 had three classes of binding sites and 1 Cd2+ bound to each binding site with Kd1 = 3.6 microM, Kd2 = 16.3 microM, Kd3 = 51.7 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human apolipoprotein A-IV binds to apolipoprotein A-I/A-II receptor sites and promotes cholesterol efflux from adipose cells

Cholesterol efflux was studied in cultured mouse adipose cells after preloading with low density lipoprotein cholesterol. Exposure to complexes containing human apolipoprotein A-IV and L-alpha-dimyristoylphosphatidylcholine (DMPC) as well as to human lipoprotein particles containing apolipoprotein A-IV but not apolipoprotein A-I and particles containing apolipoproteins A-IV and A-I showed that both artificial and native apolipoprotein A-IV-containing particles were able to promote cholesterol efflux at 37 degrees C as a function of time and concentration. The half-maximal concentration was found to be 0.3 X 10(-6) M for apolipoprotein A-IV.DMPC complexes. Binding experiments performed in intact cells at 4 degrees C with labeled apolipoprotein A-IV.DMPC complexes showed the existence of specific binding sites, with a Kd value of 0.32 x 10(-6) M and a maximal binding capacity of 223,000 sites/cell. By cross-competition experiments with labeled and unlabeled complexes containing apolipoprotein A-IV, A-I, or A-II, it appeared that all three apolipoproteins bind to the same cell-surface recognition sites. It is suggested that apolipoprotein A-IV, which is present in the interstitial fluid surrounding adipose cells in vivo at concentrations similar to those required in vitro for the promotion of cholesterol efflux, plays a critical role in cholesterol removal from peripheral cells.     

Interaction between the 35 kDa apolipoprotein of pulmonary surfactant and saturated phosphatidylcholines. Effects of temperature

We studied the interaction between the 35 kDa apolipoprotein of canine pulmonary surfactant (SP 35) and five saturated phosphatidylcholines: distearoyl (DSPC), diheptadecanoyl (DHPC), dipalmitoyl (DPPC), dimyristoyl (DMPC), and dilauroyl (DLPC); and two monoenoic unsaturated phosphatidylcholines: dioleoyl (DOPC) and dielaidyl (DEPC), using temperatures at which all of the phospholipids except DOPC were in both the gel and liquid-crystalline states. The experiments were carried out in a buffer without Ca2+. The amount of apolipoprotein which was bound by both small unilamellar and multilayered vesicles of these lipids decreased as the temperature was increased. Moreover, near the temperatures of the phase transitions of all lipids except DLPC, there was an abrupt and marked reduction in binding of protein, in that over a 3-4 degree change in temperature there was an abrupt decrease in bound apolipoprotein. A similar change in binding occurred using DLPC, although the relatively large changes in bound protein occurred at about 10 and 20 degrees C, temperatures which are above the phase transition temperature of this lipid. Experiments using DOPC were limited to temperatures above the phase transition, and apolipoprotein binding was low. Experiments monitoring the intrinsic fluorescence of the protein, and the fluorescence of bis-1-anilino-8-naphthalene sulfonic acid bound to the protein, revealed a possible conformational change at about 40 degrees C. Measurement of intrinsic fluorescence provided the same result whether or not the protein was associated with lipid. DSC of the apolipoprotein indicated that this change was not associated with a measurable thermogenic process. We found that the interaction with DPPC was reversible at 42 degrees C, and we measured the thermodynamic parameters of the interaction at this temperature. These were: delta G0 = -8.0 kcal/mol apolipoprotein; delta H0 = -88 kcal/mol; delta S0 = -254 cal/Cdeg per mol. We conclude that the interaction between SP 35 and saturated phosphatidylcholines is temperature sensitive, and this probably reflects differences in the ability of gel and liquid-crystalline phospholipids to bind this protein. Both the delta H0 and delta S0 of the interaction are negative, and may reflect an immobilization of phospholipid around the apolipoprotein to form a boundary layer. This hypothesis is consistent with the findings obtained by DSC, in which the enthalpy of the phase transition of DMPC in lipid-apolipoprotein recombinants was found to be about 60% of that expected for a pure and unperturbed multilamellar dispersion.