PubMed Health⌕ Search

Biomedical subjects

R O Ryan

Publications and source records attributed to R O Ryan.

At least 37 records · Page 2Linked to original sources
PubMed1999

Lipid binding-induced conformational changes in the N-terminal domain of human apolipoprotein E.

The N-terminal domain of human apolipoprotein E3 (apoE3) adopts an elongated, globular four helix bundle conformation in the lipid-free state. Upon lipid binding, the protein is thought to undergo a significant conformational change that is essential for manifestation of its low density lipoprotein receptor recognition properties. We have used fluorescence resonance energy transfer (FRET) to characterize helix repositioning which accompanies lipid interaction of this protein. ApoE3(1-183) possesses a single cysteine at position 112 and four tryptophan residues (positions 20, 26, 34, and 39). Modification of Cys112 with the chromophore, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)etheylenediamine (AEDANS) was specific and did not alter the secondary structure content of the protein. The efficiency of energy transfer from donor Trp residues to the AEDANS moiety was 49% in buffer, consistent with close proximity of the chromophores. Guanidine HCl titration experiments induced characteristic changes in the efficiency of energy transfer, indicating that FRET data faithfully reports on the conformational status of the protein. Interaction of AEDANS-apoE3(1-183) with dimyristoylphosphatidylcholine to form disk particles, or with detergent micelles, resulted in large decreases in the efficiency of energy transfer. Distance calculations based on the FRET measurements revealed that lipid binding increases the average distance between the four Trp donors and the AEDANS acceptor from 23 A to 44 A. The results obtained demonstrate the utility of FRET to investigate conformational adaptations of exchangeable apolipoproteins and are consistent with the hypothesis that, upon lipid binding, apoE3(1-183) undergoes conformational opening, repositioning helix 1 and 3 to adopt a receptor-active conformation.

Apolipoprotein E3↗
PubMed1998

The low density lipoprotein receptor active conformation of apolipoprotein E. Helix organization in n-terminal domain-phospholipid disc particles.

Lipid association is a prerequisite for receptor interactions of apolipoprotein E (apoE). Disc complexes of the N-terminal 22-kDa apoE3 receptor binding domain and dimyristoylphosphatidylcholine display full receptor binding activity. Studies have been performed to characterize conformational adaptations of the globular, lipid-free four-helix bundle structure that culminate in stable association of its amphipathic alpha-helices with a lipid surface. Helix-lipid interactions in bilayer disc complexes can conceivably adopt two orientations: parallel or perpendicular to the phospholipid acyl chains. Evidence based on infrared dichroism, geometrical arguments, and x-ray crystallography support the view that defined helical segments in the four-helix bundle realign upon lipid association, orienting perpendicular to the phospholipid fatty acyl chains, circumscribing the bilayer disc. Thus, it is likely that paired helical segments align in tandem, presenting a convex receptor-active surface.

Apolipoprotein E3↗
PubMed1998

Recombinant locust apolipophorin III: characterization and NMR spectroscopy.

Apolipophorin III (apoLp-III) from the locust Locusta migratoria is an exchangeable apolipoprotein that reversibly binds to lipoproteins. During lipid binding the protein has been proposed to undergo a major conformational change. To study the mechanism of lipid binding we have cloned and expressed recombinant protein in bacteria, permitting stable isotope enrichment for heteronuclear NMR spectroscopy and site-directed mutagenesis. The cDNA coding for apoLp-III was subcloned into the pET expression vector and transformed into Escherichia coli cells. Induction of expression resulted in the specific appearance of apoLp-III in the cell culture medium, indicating it escaped the bacteria without lysis. The protein was purified from the cell-free supernatant by reversed-phase HPLC, characterized and compared to the natural protein isolated from locust hemolymph. SDS-PAGE revealed the recombinant protein has a molecular mass of approximately 17 kDa, similar to that of deglycosylated natural apoLp-III. Monoclonal antibodies were used to detect recombinant apoLp-III in the cells as well as in cell-free medium of induced bacterial cultures. Amino acid sequencing and analysis confirmed the identity of the recombinant protein as L. migratoria apoLp-III. Circular dichroism spectroscopy of recombinant and natural apoLp-III showed similar spectra, both displaying high contents of alpha-helical secondary structure. Denaturation studies of lipid-free apoLp-III with guanidine hydrochloride showed that both proteins have similar denaturation midpoints and DeltaG values indicating similar protein stability. The natural and recombinant protein were functional in lipoprotein binding assays. Using recombinant protein, uniformly and specifically labeled with 15N-amino acids, two dimensional 1H-15N heteronuclear single quantum correlation spectra were obtained. The spectra revealed excellent chemical shift dispersion in both the 1H and 15N dimensions with a well defined resonance pattern. Studies with 15N-leucine specifically labeled apoLp-III in the presence and absence of the micelle forming lipid, dodecylphosphocholine, provided evidence for a significant conformational change upon lipid association.

Animals↗
PubMed1998

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.

Alkylation↗
PubMed1998

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.

Amino Acid Sequence↗
PubMed1998

Bacterial expression and characterization of chicken apolipoprotein A-I.

Apolipoprotein (apo) A-I is a 28-kDa exchangeable apolipoprotein that plays a key role in lipoprotein metabolism. It is widely distributed among animal species and is rich in alpha-helical secondary structure. Unlike human apoA-I, which aggregates in the absence of lipid, chicken apoA-I is monomeric in the lipid-free state. To take advantage of this physical characteristic, a bacterial expression system for production of recombinant chicken apoA-I has been developed. The cDNA-encoding chicken apoA-I was cloned into the pET expression vector under the regulation of the lac operon and transformed into Escherichia coli. Recombinant apoA-I protein recovered from the soluble fraction of the bacterial cell pellet was purified to greater than 95% homogeneity by reversed-phase high-performance liquid chromatography. Although immunoblot analysis confirmed the identity of the overexpressed protein, its migration on denaturing polyacrylamide gel electrophoresis was slower than its natural counterpart. To determine if the vector-encoded 18 residue pelB N-terminal leader sequence was not cleaved by the bacterial leader peptidase, isolated recombinant chicken apoA-I was incubated with exogenous leader peptidase. This treatment resulted in an increased electrophoretic mobility, with migration to a position corresponding to plasma-derived chicken apoA-I. Electrospray mass spectrometry indicated a mass of 27,961 +/- 4 Da, in agreement with that predicted for natural chicken apoA-I. Far-UV circular dichroism spectroscopy indicated an alpha-helical content similar to apoA-I isolated from chicken plasma, suggesting that the protein is folded in solution. Fluorescence studies showed that the wavelength of maximum fluorescence emission of the two tryptophan residues in the protein was 331 nm, with no shift occurring following complexation with lipid. Recombinant apoA-I was shown to be functional in lipoprotein binding as well as to possess an ability to transform bilayer vesicles of dimyristoylphosphatidylcholine into discoidal complexes. This is the first report of bacterial expression of an avian apoA-I. Increased availability and the potential for site-directed mutagenesis of this protein will aid in further characterization of apoA-I and the mechanism whereby it functions in cholesterol transport.

Animals↗
PubMed1998

Lipid transfer particle catalyzes transfer of carotenoids between lipophorins of Bombyx mori.

The yellow color of Bombyx mori hemolymph is due to the presence of carotenoids, which are primarily associated with lipophorin particles. Carotenoids were extracted from high density lipophorin (HDLp) of B. mori and analyzed by HPLC. HDLp contained 33 micrograms of carotenoids per mg protein. Over 90% of carotenoids were lutein while alpha-carotene and beta-carotene were minor components. When larval hemolymph was subjected to density gradient ultracentrifugation, a second minor yellow band was present, which was identified as B. mori lipid transfer particle (LTP). During other life stages examined however, this second band was not visible. To determine if coloration of LTP may fluctuate during development, we determined its concentration in hemolymph and compared it to that of lipophorin. Both proteins were present during all life stages and their concentrations gradually increased. The ratio of lipophorin: LTP was 10-15:1 during the fourth and fifth instar larval stages, and 20-30:1 during the pupal and adult stages. Thus, there was no correlation between the yellow color attributed to LTP and its hemolymph concentration. It is possible that yellow coloration of the LTP fraction corresponds to developmental stages when the particle is active in carotene transport. To determine if LTP is capable of facilitating carotene transfer, we took advantage of a white hemolymph B. mori strain which, when fed artificial diet containing a low carotene content, gives rise to a lipophorin that is nearly colorless. A spectrophotometric, carotene specific, transfer assay was developed which employed wild type, carotene-rich HDLp as donor particle and colorless low density lipophorin, derived from the white hemolymph strain animals, as acceptor particle. In incubations lacking LTP carotenes remained associated with HDLp while inclusion of LTP induced a redistribution of carotenes between the donor and acceptor in a time and concentration dependent manner. Time course studies suggested the rate of LTP-mediated carotene transfer was relatively slow, requiring up to 4 h to reach equilibrium. By contrast, studies employing 3H-diacylglycerol labeled HDLp as donor particle in lipid transfer assays revealed a rapid equilibration of label between the particles. Thus, it is plausible that the slower rate of LTP-mediated carotene transfer is due to its probable sequestration in the core of HDLp.

Animals↗
PubMed1998

NMR evidence for a conformational adaptation of apolipophorin III upon lipid association.

A characteristic property of amphipathic exchangeable apolipoproteins is an ability to exist alternately in lipid-free and lipid-bound states. In the present study, we have used 1H-15N-heteronuclear single quantum correlation spectroscopy to probe structural changes of apolipophorin III upon lipid association, by monitoring alterations of the chemical shifts of specific amino acids as a function of lipid titration. 15N-valine-, 15N-leucine-, 15N-lysine-, and 15N-glycine-labeled apolipophorin III were used in titration experiments with the micelle-forming lipid dodecylphosphocholine. In the absence of lipid, valine and leucine residues are located in the hydrophobic interior of the apolipophorin III helix bundle and their resonances resist chemical shift changes below the critical micelle concentration of dodecylphosphocholine. At the critical micelle concentration, however, dramatic and abrupt chemical shift changes occur, apparently coincident with formation of a protein-lipid micelle complex, as judged by significant line-width broadening of the crosspeaks. By contrast, apolipophorin III lysine and glycine residues are located on the hydrophilic surfaces of amphipathic alpha-helices or in loop regions, exposed to solvent. Their crosspeaks display either a chemical shift change similar to that seen for hydrophobic residues or a more gradual chemical shift change, beginning at very low dodecylphosphocholine concentrations. These results indicate that an interaction occurs between specific solvent-exposed lysine residues and dodecylphosphocholine below the critical micelle concentration of this lipid, whereas valine and leucine residues are not accessible to monomeric dodecylphosphocholine. At the critical micelle concentration, however, the availability of a newly formed lipid surface induces apolipophorin III binding, concomitant with conformational opening of the helix bundle, exposing its hydrophobic surfaces for binding to the dodecylphosphocholine micellar surface. Subsequently, hydrophobic residues undergo characteristic spectral changes. Subtle differences in behavior of specific hydrophobic residues, in terms of their response to dodecylphosphocholine titration and relative locations in the helix-bundle conformation, suggest that one end of the molecule may initiate contact with the lipid surface, followed by helix bundle opening.

Amino Acids↗
PubMed1997

Insight into lipid surface recognition and reversible conformational adaptations of an exchangeable apolipoprotein by multidimensional heteronuclear NMR techniques.

Apolipophorin III (apoLp-III) from the insect Manduca sexta is a 166-residue (Mr 18,340) member of the exchangeable apolipoprotein class that functions to stabilize lipid-enriched plasma lipoproteins. In the present study, we present the secondary structure and global fold of recombinant apoLp-III derived from three-dimensional heteronuclear NMR spectroscopy experiments. Five discrete alpha-helical segments (21-30 residues in length) with well defined boundaries were characterized by four NMR parameters: medium range nuclear Overhauser enhancement contacts between proton pairs, chemical shift index, coupling constants, and amide proton exchange rates. An antiparallel arrangement of helical segments has been obtained based on the long range interhelical nuclear Overhauser enhancement contacts. The NMR solution structure reveals a globular, up and down helix bundle organization similar to that of Locusta 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). However, a short helix (comprised of 5 amino acids) has been identified in the region between helix 3 and helix 4. This helix is postulated to play a role in lipid surface recognition and/or initiation of binding. Our results also indicate the existence of buried polar and charged residues in the helix bundle, providing a structural basis for the relatively low stability of apoLp-III in its lipid-free state. It is suggested that the intrinsic low stability of lipid-free apoLp-III may be important in terms of its ability to undergo a reversible, lipid binding-induced, conformational change. This study underscores the striking resemblance in molecular architecture between insect apoLp-III and the N-terminal domain of human apolipoprotein E. The potential for application of NMR techniques to studies of the exchangeable apolipoproteins, possibly in their biologically active, lipid-associated state, has broad implications in terms of our understanding of the molecular basis of their physiological functions.

Amino Acid Sequence↗
PubMed1997

Bacterial overexpression, isotope enrichment, and NMR analysis of the N-terminal domain of human apolipoprotein E.

The nucleotide sequence encoding the N-terminal domain (residues 1-183) of human apolipoprotein E3 (apoE3) was cloned into the pET expression vector and introduced into Escherichia coli. Induction of protein expression with isopropyl beta-D-thiogalactopyranoside resulted in production of recombinant apoE3(1-183). Immunoblot analysis revealed that recombinant protein was present in both the cell pellet and cell culture supernatant. Analysis revealed that a significant portion of the rApoE3(1-183) in the cell pellet still possessed the bacterial N-terminal pel B leader sequence, encoded by plasmid DNA directly upstream of the apoE3(1-183) coding sequence. By contrast, this hydrophobic leader sequence had been removed from recombinant protein specifically accumulating in the culture medium. This behavior is novel for bacterial expression of apolipoprotein E and its truncated variants and permits efficient overexpression of the recombinant protein (> 100 mg/L cell culture). Recombinant apoE3(1-183) was isolated by a combination of heparin-Sepharose chromatography and reverse-phase HPLC. Electrospray mass spectrometry provided a mass of 21 191 daltons, corresponding directly to that expected from the known sequence. Circular dichroism spectroscopy revealed that the recombinant protein possesses significant amounts of alpha-helical secondary structure. The lipid binding ability of rApoE3(1-183) was evaluated using an in vitro lipoprotein binding assay. It was observed that recombinant apoE3(1-183) protected human low density lipoprotein (LDL) from lipid accumulation induced particle aggregation, indicating that it is capable of associating with lipoprotein surfaces. In addition, rApoE3(1-183) forms disk complexes with the model phospholipid dimyristoylphosphatidylcholine. In competition experiments, it was observed that rApoE3(1-183) phospholipid disks compete with 125I-LDL for binding to the apoB/E receptor on human skin fibroblasts to an extent similar to that observed for intact rApoE3. Taken together, these data show that recombinant apoE3(1-183) is fully functional as an apolipoprotein and receptor ligand. Given the high expression level and its known existence as a monomer in solution, we evaluated the potential for application of NMR spectroscopy to study the structure-function relationship of rApoE3(1-183). Bacteria were cultured in media supplemented with 15NH4Cl or [15N]glycine and the isotopically labeled recombinant apoE3(1-183) was analyzed by heteronuclear single quantum correlation NMR spectroscopy. The data revealed that rApoE3(1-183) is an excellent candidate for solution structure studies by NMR, including conformational adaptations associated with lipid association.

Apolipoprotein E3↗
PubMed1996

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.

Animals↗
PubMed1996

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.

Animals↗
PubMed1996

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.

Amides↗
PubMed1996

Merck Frosst award lecture 1995. La conference Merck Frosst 1995. Structural studies of lipoproteins and their apolipoprotein components.

Lipid transport processes via the circulatory system of animals are a vital function that utilizes highly specialized lipoprotein complexes. These complexes of protein and lipid impart solubility to otherwise insoluble lipids. The apoprotein components of lipoprotein complexes serve to stabilize the lipid components and modulate particle metabolism and function as ligands for receptor-mediated endocytosis of lipoproteins. We have used an insect (Manduca sexta) model system for studies of lipid transport. In this system, flight activity elicits a dramatic increase in the demand for glycerolipid fuel molecules by flight muscle tissue. These lipids are mobilized from a storage organ and transported through the hemolymph (blood) to the flight muscle by the lipoprotein, lipophorin. This system possesses the unique property that lipids are loaded onto pre-existing high density lipophorin through the action of a lipid transfer particle (LTP). LTP is a high molecular weight hemolymph component that facilitates net vectorial lipid transfer from fat body tissue to lipophorin. The increase in lipid content of the lipoprotein induces association of a low molecular weight amphipathic exchangeable apolipoprotein, apolipophorin III (apoLp-III). ApoLp-III is a 18 kDa protein that normally exists as a water-soluble monomeric hemolymph protein. The structural properties of apoLp-III have been investigated by X-ray crystallography. ApoLp-III from Locusta migratoria adopts a five helix bundle conformation wherein each of the amphipathic helices orients with its hydrophobic face directed toward the interior of the bundle. It has been hypothesized that lipid association requires a dramatic conformational change wherein the helix bundle opens about putative hinge domains located in the loops between helices. The data accumulated support the concept that apoLp-III is a member of the broad class of exchangeable apolipoproteins and structural information learned from this system is directly applicable to analogous proteins in higher organisms.

Animals↗
PubMed1995

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)

Amino Acid Sequence↗
PubMed1995

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.

Animals↗
PubMed1995

Identification and localization of two distinct microenvironments for the diacylglycerol component of lipophorin particles by 13C NMR.

13C nuclear magnetic resonance spectroscopy of lipoproteins, isolated from the insect Manduca sexta, has been employed to probe the microenvironment of diacylglycerol (DG), their major neutral lipid component. Natural abundance 13C NMR spectra of high density lipophorin exhibited several well-separated resonances derived from its lipid moiety, including those for the carbonyl carbon atoms of phospholipid and DG fatty acyl chains in the region of 175-180 ppm. To verify the assignment of the DG acyl chain carbonyl carbon resonances, di[1-13C]oleoylglycerol high density lipophorin was isolated after instilling a bolus of tri[1-13C]oleoylglycerol into the midgut of larvae fed a fat-free diet. 13C NMR spectra of the isolated lipoprotein revealed a specific and dramatic enrichment of resonances at 175.5 ppm. Expansion of this region revealed two resonances separated by 0.08 ppm. These were assigned as 1,2- and 1,3- isomers of DG, the latter presumably arising from spontaneous acyl chain migration of 1,2-DG following lipoprotein isolation. On the basis of compositional and structural analysis of this lipoprotein, it is postulated that these DG species are localized predominantly in the hydrophobic core of the particle. By contrast, natural abundance 13C NMR spectra of the DG-rich, low density lipophorin (LDLp) subspecies revealed two additional resonances, separated by 0.2 ppm, that were tentatively assigned as 1,2- and 1,3-DG present at the surface of the particle. The verify this assignment, experiments employing phospholipase C, to convert lipophorin surface associated phospholipid into DG, were performed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗