The LDL receptor binding domain of apolipoprotein E directs the relative orientation of its C-terminal segment in reconstituted nascent HDL.

Apolipoprotein E (apoE) (299 residues) is a highly helical protein that plays a critical role in cholesterol homeostasis. It comprises a four-helix bundle N-terminal (NT) and a C-terminal (CT) domain that can exist in lipid-free and lipid-associated states. In humans, there are two major apoE isoforms, apoE3 and apoE4, which differ in a single residue in the NT domain, with apoE4 strongly increasing risk of Alzheimer's disease (AD) and cardiovascular diseases (CVD). It has been proposed that the CT domain initiates rapid lipid binding, followed by a slower NT domain helix bundle opening and lipid binding to yield discoidal reconstituted high density lipoprotein (rHDL). However, the contribution of the NT domain on the CT domain organization in HDL remains poorly understood. To understand this, we employed Cys-specific cross-linking and spatially-sensitive fluorophores in the NT and CT domains of apoE3 and apoE4, and in isolated CT domain. We noted that the helices in isolated CT domain are oriented parallel to those in the neighboring molecule in rHDL, whereas full length apoE3 and apoE4 adopt either an anti-parallel or hairpin-like organization. It appears that the bulky NT domain determines the spatial organization of its CT domain in rHDL, a finding that has significance for apoE4, which is more susceptible to proteolytic cleavage in AD brains, showing increased accumulation of neurotoxic NT and CT fragments. We envisage that the structural organization of HDL apoE would have profound functional consequences in its ability to regulate cholesterol homeostasis in AD and CVD.

Diet-induced obesity: dopamine transporter function, impulsivity and motivation.

OBJECTIVE: A rat model of diet-induced obesity (DIO) was used to determine dopamine transporter (DAT) function, impulsivity and motivation as neurobehavioral outcomes and predictors of obesity. DESIGN: To evaluate neurobehavioral alterations following the development of DIO induced by an 8-week high-fat diet (HF) exposure, striatal D2-receptor density, DAT function and expression, extracellular dopamine concentrations, impulsivity, and motivation for high- and low-fat reinforcers were determined. To determine predictors of DIO, neurobehavioral antecedents including impulsivity, motivation for high-fat reinforcers, DAT function and extracellular dopamine were evaluated before the 8-week HF exposure. METHODS: Striatal D2-receptor density was determined by in vitro kinetic analysis of [(3)H]raclopride binding. DAT function was determined using in vitro kinetic analysis of [(3)H]dopamine uptake, methamphetamine-evoked [(3)H]dopamine overflow and no-net flux in vivo microdialysis. DAT cell-surface expression was determined using biotinylation and western blotting. Impulsivity and food-motivated behavior were determined using a delay discounting task and progressive ratio schedule, respectively. RESULTS: Relative to obesity-resistant (OR) rats, obesity-prone (OP) rats exhibited 18% greater body weight following an 8-week HF-diet exposure, 42% lower striatal D2-receptor density, 30% lower total DAT expression, 40% lower in vitro and in vivo DAT function, 45% greater extracellular dopamine and twofold greater methamphetamine-evoked [(3)H]dopamine overflow. OP rats exhibited higher motivation for food, and surprisingly, were less impulsive relative to OR rats. Impulsivity, in vivo DAT function and extracellular dopamine concentration did not predict DIO. Importantly, motivation for high-fat reinforcers predicted the development of DIO. CONCLUSION: Human studies are limited by their ability to determine if impulsivity, motivation and DAT function are causes or consequences of DIO. The current animal model shows that motivation for high-fat food, but not impulsive behavior, predicts the development of obesity, whereas decreases in striatal DAT function are exhibited only after the development of obesity.

Full-length apolipoprotein E protects against the neurotoxicity of an apoE-related peptide.

Apolipoprotein E was found to protect against the neurotoxic effects of a dimeric peptide derived from the receptor-binding region of this protein (residues 141-149). Both apoE3 and apoE4 conferred protection but the major N-terminal fragment of each isoform did not. Nor was significant protection provided by bovine serum albumin or apoA-I. Full-length apoE3 and apoE4 also inhibited the uptake of a fluorescent-labeled derivative of the peptide, suggesting that the mechanism of inhibition might involve competition for cell surface receptors/proteoglycans that mediate endocytosis and/or signaling pathways. These results might bear on the question of the role of apoE in neuronal degeneration, such as occurs in Alzheimer's disease where apoE4 confers a significantly greater risk of pathology.

Lipid association-induced N- and C-terminal domain reorganization in human apolipoprotein E3.

Apolipoprotein E (apoE) is a 299 amino acid, anti-atherogenic protein that plays a key role in regulating plasma lipoprotein metabolism. It is composed of an N-terminal (NT) domain (residues 1-191) that is responsible for binding to members of the low density lipoprotein receptor family and a C-terminal (CT) domain (residues 216-299) that anchors the protein to lipoprotein particles by virtue of its high-affinity lipid binding characteristics. Isoform-specific differences in the NT domain that modulate the lipoprotein binding preference elicited by the CT domain suggest the existence and importance of domain interactions in this protein. Employing steady state fluorescence quenching and resonance energy transfer techniques, spatial proximity relationships between the N- and C-terminal domains were investigated in recombinant human apoE3. ApoE3 containing a single Trp at position 264 and an N-iodoacetyl-N'-(5-sulfo-1-napthyl) ethylenediamine (AEDANS) moiety covalently attached to the lone Cys residue at position 112 was used (AEDANS-apoE3/W@264). Fluorescence quenching studies revealed a solvent-exposed location for Trp-264. In the lipid-free state, fluorescence resonance energy transfer (FRET) was noted between Trp-264 and AEDANS, with a calculated distance of 27 A between the two fluorophores. Control experiments established that FRET observed in this system is intramolecular. FRET was abolished upon proteolysis in the linker region connecting the NT and CT domains. Lowering the solution pH to 4 induced an increase in the efficiency of intramolecular energy transfer, with the two domains reorienting about 5 A closer to one another. Interdomain FRET was retained in the presence of 0.6-1.0 m guanidine hydrochloride but was lost at higher concentrations, a manifestation of unfolding of the domains and increased distance between the donor-acceptor pair. Interaction of AEDANS-apoE3/W@264 with lipid induced a loss of FRET, attributed to spatial repositioning of the domains by >80 A. The data provide biophysical evidence that, in addition to reported conformational changes in the four-helix bundle configuration induced by lipid association, lipid binding of apoE is accompanied by reorientation of the tertiary disposition of the NT and CT domains.

Modulation of the lipid binding properties of the N-terminal domain of human apolipoprotein E3.

Apolipoprotein E (apoE) plays a critical role in plasma lipid homeostasis through its function as a ligand for the low-density lipoprotein (LDL) receptor family. Receptor recognition is mediated by residues 130-150 in the independently folded, 22-kDa N-terminal (NT) domain. This elongated globular four-helix bundle undergoes a conformational change upon interaction with an appropriate lipid surface. Unlike other apolipoproteins, apoE3 NT failed to fully protect human LDL from aggregation induced by treatment with phospholipase C. Likewise, in dimyristoylglycerophosphocholine (Myr2Gro-PCho) vesicle transformation assays, 100 microg apoE3 NT induced only 15% reduction in vesicle (250 microg) light scattering intensity after 30 min. ApoE3 NT interaction with modified lipoprotein particles or Myr2Gro-PCho vesicles was concentration-dependent whereas the vesicle transformation reaction was unaffected by buffer ionic strength. In studies with the anionic phospholipid dimyristoylglycerophosphoglycerol, apoE3 NT-mediated vesicle transformation rates were enhanced > 10-fold compared with Myr2Gro-PCho and activity decreased with increasing buffer ionic strength. Solution pH had a dramatic effect on the kinetics of apoE3 NT-mediated Myr2Gro-PCho vesicle transformation with increased rates observed as a function of decreasing pH. Fluorescence studies with a single tryptophan containing apoE3 NT mutant (L155W) revealed increased solvent exposure of the protein interior at pH values below 4.0. Similarly, fluorescent dye binding experiments with 8-anilino-1-naphthalene sulfonate revealed increased exposure of apoE3 NT hydrophobic interior as a function of decreasing pH. These studies indicate that apoE3 NT lipid binding activity is modulated by lipid surface properties and protein tertiary structure.

The lipid-associated conformation of the low density lipoprotein receptor binding domain of human apolipoprotein E.

Apolipoprotein E (apoE) is a 34-kDa exchangeable apolipoprotein that regulates metabolism of plasma lipoproteins by functioning as a ligand for members of the LDL receptor family. The receptor-binding region localizes to the vicinity of residues 130-150 within its independently folded 22-kDa N-terminal domain. In the absence of lipid, this domain exists as a receptor-inactive, globular four-helix bundle. Receptor recognition properties of this domain are manifest upon lipid association, which is accompanied by a conformational change in the protein. Fluorescence resonance energy transfer has been used to monitor helix repositioning, which accompanies lipid association of the apoE N-terminal domain. Site-directed mutagenesis was used to replace naturally occurring Trp residues with phenylalanine, creating a Trp-null apoE3 N-terminal domain (residues 1-183). Subsequently, tyrosine residues in helix 2, helix 3, or helix 4 were converted to Trp, generating single Trp mutant proteins. The lone cysteine at position 112 was covalently modified with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine, which serves as an energy acceptor from excited tryptophan residues. Fluorescence resonance energy transfer analysis of apoE N-terminal domain variants in phospholipid disc complexes suggests that the helix bundle opens to adopt a partially extended conformation. A model is presented that depicts a tandem arrangement of the receptor-binding region of the protein in the disc complex, corresponding to its low density lipoprotein receptor-active conformation.

Pyrene excimer fluorescence: a spatially sensitive probe to monitor lipid-induced helical rearrangement of apolipophorin III.

Manduca sexta apolipophorin III (apoLp-III), an 18-kDa, monomeric, insect hemolymph apolipoprotein, is comprised of five amphipathic alpha-helices arranged as a globular bundle in the lipid-free state. Upon lipid binding, it is postulated that the bundle opens, exposing a continuous hydrophobic surface which becomes available for lipid interaction. To investigate lipid binding-induced helical rearrangements, we exploited the unique fluorescence characteristics of N-(1-pyrene)maleimide. Pyrene is a spatially sensitive extrinsic fluorescent probe, which forms excited-state dimers (excimers) upon close encounter with another pyrene molecule. Cysteine residues were introduced into apoLp-III (which otherwise lacks cysteine) at Asn 40 (helix 2) and/or Leu 90 (helix 3), creating two single-cysteine mutants (N40C-apoLp-III and L90C-apoLp-III) and N40C/L90C-apoLp-III, a double-cysteine mutant, which were labeled with pyrene maleimide. Pyrene-labeled N40C/L90C-apoLp-III, but not the pyrene-labeled single-cysteine mutants, exhibited strong excimer fluorescence in the lipid-free, monomeric state. Guanidine hydrochloride titration and temperature studies revealed a loss in excimer fluorescence, accompanied by a loss in the molar ellipticity of the protein. When apoLp-III interacts with phospholipid vesicles to form disklike complexes, a significant loss in excimer fluorescence was noted, indicating that the helices bearing the pyrene moieties diverge from each other. Pyrene excimer fluorescence was further employed to examine the relative orientation of lipid-bound apoLp-III molecules. Pyrene-labeled N40C- or L90C-apoLp-III displayed no excimer fluorescence in the disk complexes, while complexes prepared with an equal mixture of both single-labeled mutants did emit excimer fluorescence, indicating apoLp-III adopts a preferred nonrandom orientation around the perimeter of the bilayer disk. These studies establish pyrene excimer fluorescence as a useful spectroscopic tool to address intra- and intermolecular interactions of exchangeable apolipoproteins upon binding to lipid.

Spectroscopic characterization of the conformational adaptability of Bombyx mori apolipophorin III.

Apolipophorin III (apoLp-III) from the silkmoth, Bombyx mori, has been over-expressed in Escherichia coli, purified and characterized. Far-UV CD spectroscopic analysis revealed 65% alpha-helix secondary structure. Near-UV CD spectra obtained in buffer or complexed with dimyristoylglycerophosphocholine (DMPC), provided evidence that apoLp-III alpha-helices reorient upon interaction with lipid, indicative of a protein conformational change. In guanidine hydrochloride (GdnHCl) denaturation studies, a transition midpoint of 0.33 M was observed, corresponding to a DeltaGDH2O = 2.46 kcal. mol-1. Fluorescence studies of the sole tryptophan residue (Trp40) in apoLp-III revealed an emission lambdamax = 327 nm. Compared to free tryptophan, Stern-Volmer constants (KSV) for acrylamide and KI quenching of Trp40 fluorescence were decreased by 20-fold and sevenfold, respectively. In studies of apoLp-III-DMPC disc complexes, far-UV CD spectroscopy revealed an increase in alpha-helix content to approximately 85% and a ninefold increase in the GdnHCl-induced denaturation transition midpoint to 3 M. In studies of lipid interaction, apoLp-III was shown to disrupt both negatively charged and zwitterionic phospholipid bilayer vesicles, transforming them into discoidal complexes. Characterization of apoLp-III-DMPC discs, using 5-doxyl or 12-doxyl stearic acid as lipid-based quenching agents, revealed that Trp40 localizes near the phospholipid polar head groups. KSV values for acrylamide and KI quenching of intrinsic fluorescence of apoLp-III-DMPC discs indicate that Trp40 is embedded in the lipid milieu, with little or no accessibility to the aqueous quenchers. Given the large amount of alpha-helix in apoLp-III, the data presented support a model in which amphipathic alpha-helical segments are stabilized by helix-helix interactions and lipid association induces a protein conformational change which results in substitution of helix-helix interactions for helix-lipid contacts.

Interaction of an exchangeable apolipoprotein with phospholipid vesicles and lipoprotein particles. Role of leucines 32, 34, and 95 in Locusta migratoria apolipophorin III.

Apolipophorin III (apoLp-III) from Locusta migratoria is an exchangeable apolipoprotein that binds reversibly to lipid surfaces. In the lipid-free state this 164-residue protein exists as a bundle of five elongated amphipathic alpha-helices. Upon lipid binding, apoLp-III undergoes a significant conformational change, resulting in exposure of its hydrophobic interior to the lipid environment. On the basis of x-ray crystallographic data (Breiter, D. R., Kanost, M. R., Benning, M. M., Wesenberg, G., Law, J. H., Wells, M. A., Rayment, I., and Holden, H. M. (1991) Biochemistry 30, 603-608), it was proposed that hydrophobic residues, present in loops that connect helices 1 and 2 (Leu-32 and Leu-34) and helices 3 and 4 (Leu-95), may function in initiation of lipid binding. To examine this hypothesis, mutant apoLp-IIIs were designed wherein the three Leu residues were replaced by Arg, individually or together. Circular dichroism spectroscopy and temperature and guanidine hydrochloride denaturation studies showed that the mutations did not cause major changes in secondary structure content or stability. In lipid binding assays, addition of apoLp-III to phospholipid vesicles caused a rapid clearance of vesicle turbidity due to transformation to discoidal complexes. L34R and L32R/L34R/L95R apoLp-IIIs displayed a much stronger interaction with lipid vesicles than wild-type apoLp-III. Furthermore, it was demonstrated that the mutant apoLp-IIIs retained their ability to bind to lipoprotein particles. However, in lipoprotein competition binding assays, the mutants displayed an impaired ability to initiate a binding interaction when compared with wild-type apoLp-III. The data indicate that the loops connecting helices 1 and 2 and helices 3 and 4 are critical regions in the protein, contributing to recognition of hydrophobic defects on lipoprotein surfaces by apoLp-III.

A molecular trigger of lipid binding-induced opening of a helix bundle exchangeable apolipoprotein.

Apolipophorin III (apoLp-III) from the sphinx moth, Manduca sexta, is a helix bundle protein that interacts reversibly with lipoproteins. Its five elongated amphipathic alpha-helices are organized in an antiparallel fashion, with helices 3 and 4 connected by a short 6-residue (PDVEKE) linker helix, termed helix 3'. Upon interaction with lipoproteins, apoLp-III opens to expose a continuous hydrophobic interior. It was postulated that helix bundle opening is preceded by an initiation step wherein helix 3' serves to recognize available lipoprotein surface binding sites. To test this hypothesis, helix 3' was replaced by residues that have a propensity to form a type I beta-turn, NPNG. This mutant apoLp-III was defective in lipoprotein binding assays. To define a more precise mode of interaction, the relevance of the presence of the hydrophobic Val-97 flanked by Asp-96 and Glu-98 was investigated by site-directed mutagenesis. V97N and D96N/V97N/E98Q apoLp-III were unable to compete with wild-type apoLp-III to initiate an interaction with lipoproteins, whereas D96N/E98Q apoLp-III was as competent as wild-type apoLp-III. The results suggest that Val-97 is critical, whereas Asp-96 and Glu-98 are irrelevant for initiating binding to lipoproteins. A model of binding is presented wherein apoLp-III is oriented with the helix 3' end of the molecule juxtaposed to the lipoprotein surface. Recognition of lipoprotein surface hydrophobic defects by Val-97 triggers opening of the helix bundle and facilitates formation of a stable binding interaction.

Interhelical contacts are required for the helix bundle fold of apolipophorin III and its ability to interact with lipoproteins.

Apolipophorin-III (apoLp-III) from the insect, Manduca sexta, is a 166-residue exchangeable apolipoprotein that plays a critical role in the dynamics of plasma lipoprotein interconversions. Our previous work indicated that a 36-residue C-terminal peptide fragment, generated by cyanogen bromide digestion of apoLp-III, was unable to bind to lipid surfaces (Narayanaswami V, Kay CM, Oikawa K, Ryan RO, 1994, Biochemistry 33:13312-13320), and showed no secondary structure in aqueous solution. In this paper, we have performed structural studies of this peptide (E131-Q166) complexed with SDS detergent micelles, or in the presence of the helix-inducing solvent trifluoroethanol (TFE), by two-dimensional 1H NMR spectroscopy. The peptide adopts an alpha-helical structure in the presence of both SDS and 50% TFE. The lipid-bound structure of the peptide, generated from the NMR NOE data, showed an elongated, slightly curved alpha-helix. Despite its high alpha-helix forming propensity, the peptide requires alpha helix-promoting environment to adopt an alpha-helical structure. This indicates the importance of the surrounding chemical environment and implies that, in the absence of lipid, tertiary contacts in the folded protein play a role in maintaining its structural integrity. Furthermore, the data suggest that the amphipathic helix bundle organization serves as a prerequisite structural motif for the reversible lipoprotein-binding activity of M. sexta apoLp-III.

Fluorescence studies of exchangeable apolipoprotein-lipid interactions. Superficial association of apolipophorin III with lipoprotein surfaces.

Apolipophorin III (apoLp-III) from the Sphinx moth, Manduca sexta, is an 18-kDa exchangeable apolipoprotein that reversibly associates with lipoprotein particles. In the absence of lipid, apoLp-III exists as an elongated bundle of five amphipathic alpha-helices. Upon lipid association, the protein is postulated to undergo a major conformational change, wherein the bundle opens around hinge loop regions, resulting in exposure of its hydrophobic interior. Fluorescence quenching techniques have been employed to study apoLp-III helix topography and spatial arrangement in phospholipid disc complexes and intact lipoprotein particles. Intrinsic fluorescence of the single tyrosine in apoLp-III was exploited to monitor the location of helix 5 in model disc complexes. To investigate other regions of the protein, site-directed mutagenesis was performed to introduce cysteine residues, replacing Asn-40 (helix 2, N40C) or Leu-90 (helix 3, L90C), thereby providing two mutant apoLp-IIIs, each with a single site for covalent attachment of the extrinsic fluorescent probe, N-(1-pyrene) maleimide. In the lipid-free state, pyrene-N40C- and pyrene-L90C-apoLp-III were highly accessible to the negatively charged aqueous quencher KI, yielding Ksv values of 27.1 and 19.8 M-1, respectively. Upon binding to the surface of a spherical lipoprotein particle, Ksv values for KI decreased by about 90% for both pyrene-labeled apoLp-IIIs, indicating a significant change in the local microenvironment of the fluorophores. A lesser decrease in Ksv was observed when the pyrene-labeled apoLp-IIIs were bound to phospholipid disc complexes. When spin-labeled fatty acids 5-doxylstearic acid and 12-doxylstearic acid were used as lipophilic quenchers, tyrosine and pyrene fluorescence were more effectively quenched by 5-doxylstearic acid in both phospholipid bilayer disc complexes and spherical lipoprotein particles. These data provide insight into the spatial topography of apoLp-III alpha-helices in phospholipid disc complexes and support the concept that interaction with spherical lipoprotein particles results in superficial contact of apoL-III helical segments with the monolayer surface, providing a basis for its reversible binding ability.

Fluorescence studies of lipid association-induced conformational adaptations of an exchangeable amphipathic apolipoprotein.

The conformational adaptability of Manduca sexta apolipophorin III (apoLp-III) has been evaluated by monitoring the spectroscopic properties of its sole tyrosine residue, Tyr145, present in the fifth helical segment of the protein. M. sexta apoLp-III adopts a globular five-helix bundle structure in solution and has been postulated to undergo an opening at putative hinge domains upon interaction with lipid surfaces. Previous results have shown that the intrinsic fluorescence of Tyr145 is highly quenched in the closed, water-soluble conformation but is dramatically enhanced upon lipid association. We have carried out a spectroscopic characterization of Tyr145 and its microenvironment, to enable its use as a structural probe of lipid-induced conformational changes of apoLp-III. The pKa of Tyr145 in lipid-free apoLp-III was found to be 10.5, as determined from uv-spectrophotometry, indicating that, in the ground state, the tyrosyl phenolic group is not ionized under physiological conditions. Compared to free tyrosine in aqueous buffer (pH 7.0), a red shift (77 nm) in the (lambda)max of absorbance of Tyr145 was observed, suggesting that an H-bonding interaction is responsible for the quenched state of tyrosine fluorescence. In an effort to explain the observed quenching phenomenon, the quantum yield and lifetimes of Tyr145 fluorescence emission were investigated as a function of pH and lipid binding. The quantum yield of Tyr145 in lipid-free apoLp-III was enhanced fivefold upon decreasing the pH, with a half-maximal point around pH 5.5. Time-resolved fluorescence decay analysis showed that Tyr145 exhibits nonexponential emission decay with two components having lifetimes of 3.3 ns (76%) and 0.89 ns (24%) in the lipid-free state. The lifetime and amplitude of Tyr145 remain essentially unaltered upon lipid association or decreasing the pH. This is consistent with the hypothesis that, in the lipid-free helix bundle conformation, a quenching residue exists within H-bonding distance of the phenolic side chain of Tyr145 which, at physiological pH, is responsible for the observed fluorescence quenching. Opening of the helix bundle repositions this acceptor base, possibly a carboxylate or an imidazole side chain, making it unavailable for quenching. Using differential polarized phase and modulation fluorometry, it was seen that the segmental motion of Tyr145 is also altered considerably upon lipid interaction. These spectroscopic and motional properties of Tyr145 distinguish this unique residue as a useful probe to monitor structural flexibility of apoLp-III.

Disulfide bond engineering to monitor conformational opening of apolipophorin III during lipid binding.

Apolipophorin III (apoLp-III) from the Sphinx moth, Manduca sexta, is an exchangeable, amphipathic apolipoprotein that alternately exists in water-soluble and lipid-bound forms. It is organized as a five-helix bundle in solution, which has been postulated to open at putative hinge domains to expose the hydrophobic interior, thereby facilitating interaction with the lipoprotein surface (Breiter, D. R. , Kanost, M. R., Benning, M. M., Wesenberg, G., Law, J. H., Wells, M. A., Rayment, I., and Holden, H. M. (1991) Biochemistry 30, 603-608). To test this hypothesis, we engineered two cysteine residues in apoLp-III, which otherwise lacks cysteine, by site-directed mutagenesis at Asn-40 and Leu-90. Under oxidizing conditions the two cysteines spontaneously form a disulfide bond, which should tether the helix bundle and thereby prevent opening and concomitant lipid interaction. N40C/L90C apoLp-III was overexpressed in Escherichia coli and characterized for disulfide bond formation, secondary structure content, and stability, under both oxidizing and reducing conditions. Functional characterization was carried out by comparing the abilities of the oxidized and reduced protein to associate with modified lipoproteins in vitro. While the reduced form behaved like wild type apoLp-III, the oxidized form was unable to associate with lipoproteins. These results suggest that opening of the helix bundle is required for interaction with lipoproteins and provide a molecular basis for the dual existence of water-soluble and lipid-bound forms of apoLp-III. However, in phospholipid bilayer association assays, wild type, reduced, and oxidized N40C/L90C apoLp-III exhibited similar abilities to transform dimyristoylphosphatidylcholine multilamellar vesicles to disc-like complexes, as judged by electron microscopy. These data emphasize that underlying differences exist in initiating or maintaining a stable interaction of apoLp-III with phospholipid disc complexes versus spherical lipoprotein surfaces.

Hydrogen/deuterium exchange kinetics of apolipophorin-III in lipid-free and phospholipid-bound states. An analysis by Fourier transform infrared spectroscopy.

Attenuated total reflection Fourier transform infrared spectroscopy was used to probe the kinetics of hydrogen/deuterium exchange in Manduca sexta apolipophorin-III (apoLp-III). ApoLp-III is an exchangeable apolipoprotein that is made up of five elongated amphipathic alpha-helices in a helical bundle conformation in the monomeric lipid-free form. Upon interaction with phospholipids, it is postulated to undergo a large conformational change whereby the hydrophobic interior is exposed, facilitating binding to the lipid surfaces. We have used the lipid-free and dimyristoylphosphatidylcholine-bound apoLp-III to study the dynamically variable domains in the two forms. Three populations of amide protons varying in their hydrogen/deuterium exchange rates were found to exist: slow, intermediate, and fast exchanging, which could correspond to completely buried, partially buried, and solvent-exposed domains on the protein in both the states. In lipid-free apoLp-III, 36, 12, and 52% of the total residues contributed to the slow, intermediate, and fast exchanging populations, respectively. In the dimyristoylphosphatidylcholine-bound form, the corresponding distribution was 20, 16, and 64%, representing a 12% increase in the number of exposed residues. The results are discussed in terms of increased solvent accessibility due to gross tertiary structural reorganization.

Spectroscopic and lipid binding studies on the amino and carboxyl terminal fragments of Locusta migratoria apolipophorin III.

The structural basis for the lipid binding capability of Locusta migratoria apolipophorin III (apoLp-III) was assessed by characterizing the amino and carboxyl terminal halves of the protein. The native molecule (approximately 20 kDa) was deglycosylated with endoglycosidase F (molecular mass of deglycosylated species approximately 18 kDa) and cleaved with endoproteinase Arg-C to yield two fragments with molecular masses of approximately 9 kDa each. The two fragments were purified by reversed-phase HPLC and identified by mass spectrometry, amino acid analysis and N-terminal sequencing as the amino terminal (N9) and carboxyl terminal (C9) halves. Due to the apparent discrepancy of the protease digestion pattern obtained compared to that expected from the deduced amino sequence of apoLp-III cDNA, we carried out partial amino acid sequencing of the fragments and cDNA sequencing for the entire protein. Circular dichroism spectroscopy of the N9 and C9 peptides revealed that both exist in buffer in a random coil state. However, addition of trifluoroethanol, a helix-inducing agent, resulted in the formation of an alpha-helix, reflecting an innate propensity of the peptides to adopt a helical conformation. When cosonicated with dimyristoylphosphatidylcholine (DMPC) both peptides assumed an alpha-helical conformation, indicative of interaction with the phospholipid. In the presence of phospholipids, a 22 nm blue shift in Trp fluorescence emission was observed in the case of the C9 peptide, suggesting that the Trp residues are located in a more hydrophobic environment. Electron microscopy revealed that, compared to native apoLp-III, both peptides possessed a reduced ability to transform DMPC vesicles to disklike complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Alignment of the apolipophorin-III alpha-helices in complex with dimyristoylphosphatidylcholine. A unique spatial orientation.

Apolipophorin-III (apoLp-III) from Manduca sexta can exist in two alternate states: as a globular, lipid-free helix bundle or a lipid surface-associated apolipoprotein. Previous papers (Ryan R.O., Oikawa K., and Kay C. M. (1993) J. Biol. Chem. 268, 1525-1530; Wientzek M., Kay C.M., Oikawa K., and Ryan R.O. (1994) J. Biol. Chem. 269, 4605-4612) have investigated the structures and properties of apolipophorin-III from M. sexta in the lipid-free state and associated to lipids. Association of apoLp-III with dimyristoylphosphatidylcholine vesicles leads to the formation of uniform lipid discs with an average diameter and thickness of 18.5 +/- 2.0 and 4.8 +/- 0.8 nm, respectively. These discs contain six molecules of apoLp-III. Geometrical calculations based on these data, together with x-ray crystallographic data from the homologous L. migratoria apoLp-III (Breiter D. R., Kanost M.R., Benning M.M., Wesenberg G., Law J.H., Wells M.A., Rayment I., and Holden H.M. (1991) Biochemistry 30, 603-608), have allowed the presentation of a model of lipid-protein interaction, in which the alpha-helices of the apoLp-III orient perpendicular to the phospholipid chains and surround the lipid disc. Here, using polarized Fourier transform-attenuated total reflection infrared spectroscopy, we provide the first experimental evidence of a unique perpendicular orientation of the alpha-helices with respect to the fatty acyl chains of the phospholipids in the disc.

Bacterial expression and site-directed mutagenesis of a functional recombinant apolipoprotein.

To facilitate structure-function studies of Manduca sexta apolipophorin III (apoLp-III), its nucleotide coding sequence was cloned from a fat body cDNA library by in vitro DNA amplification. The amplification product was cloned in the pET expression vector and introduced into E. coli. After induction, cultures were screened for apoLp-III protein production by immunoblotting with anti-apoLp-III serum. Data obtained indicated the presence of apoLp-III in both cell lysates and media of cell cultures harboring the apoLp-III-pET construct but not in cells containing the parent vector. The protein was isolated from the cell-free supernatant of cultures grown in minimal media 4 h after induction. Verification that the recombinant protein produced was indeed apoLp-III was obtained by electrospray mass spectrometric analysis. Circular dichroism (CD) spectroscopy of the isolated recombinant protein revealed a characteristic content of alpha-helical secondary structure with a further induction of helix upon addition of 50% trifluoroethanol. In urea denaturation studies, monitored by CD, evidence was obtained that recombinant and natural apoLp-III possess indistinguishable thermodynamic properties. In addition, lipid binding assays revealed that recombinant apoLp-III formed stable complexes with phospholipids and was capable of associating with lipoprotein surfaces. Examination of the fluorescence properties of recombinant apoLp-III revealed the presence of a noncovalently associated fluorescent contaminant that was effectively removed by reverse phase HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)