Structural and Binding Characteristics of the Carboxyl Terminal Fragment of Apolipophorin III from Manduca sexta

Narayanaswami, Vasanthy; Kay, Cyril M.; Oikawa, Kimio; Ryan, Robert O.

doi:10.1021/bi00249a018

Cited by 20 publications

(24 citation statements)

References 51 publications

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“…Cleavage of L. migratoria apoLp-III in the middle of helix-3 yielded two 9K fragments, but those fragments failed to interact spontaneously with DMPC vesicles (21). Isolation of a 4K fragment of M. sexta apoLp-III that consists of the fifth helix displayed a predominantly random-coiled, unstructured state (20), and did not bind to DMPC vesicles. From NMR studies with the 4K peptide (22) as well as with full-length protein (13), it was concluded that hydrophobic interhelical contacts, such as between helix-1 and helix-5 and between helix-2 and helix-5, are necessary for proper folding of apoLp-III.…”

Section: Structural Studies With Apolp-iiimentioning

confidence: 98%

An N-Terminal Three-Helix Fragment of the Exchangeable Insect Apolipoprotein Apolipophorin III Conserves the Lipid Binding Properties of Wild-Type Protein

et al. 2001

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

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Section: Structural Studies With Apolp-iiimentioning

confidence: 98%

An N-Terminal Three-Helix Fragment of the Exchangeable Insect Apolipoprotein Apolipophorin III Conserves the Lipid Binding Properties of Wild-Type Protein

et al. 2001

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“…In earlier studies, methionine has been used successfully to produce an apoLp-III deletion variant and a helix fragment by cyanogen bromide digestion. This was done by employing the single methionine already present in Manduca sexta apoLp-III (36) or by introduction of a methionine in the L. migratoria helix bundle (23). Nevertheless, alternative strategies to remove the carrier protein, such as inclusion of a target sequence for enzyme-mediated proteolysis, can be easily implemented as custom made nucleotide synthesis, which include codon optimization, has become relatively inexpensive.…”

Section: Discussionmentioning

confidence: 99%

Expression of the C-terminal domain of human apolipoprotein A-I using a chimeric apolipoprotein

Sallee

Horn

Fuentes

et al. 2017

Protein Expression and Purification

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Human apolipoprotein A-I (apoA-I) is the most abundant protein in high-density lipoprotein, an anti-atherogenic lipid-protein complex responsible for reverse cholesterol transport. The protein is composed of an N-terminal helix bundle domain, and a small C-terminal (CT) domain. To facilitate study of CT-apoA-I, a novel strategy was employed to produce this small domain in a bacterial expression system. A protein construct was designed of insect apolipophorin III (apoLp-III) and residues 179–243 of apoA-I, with a unique a methionine residue positioned between the two proteins and an N-terminal His-tag to facilitate purification. The chimera was expressed in E. coli, purified by Ni-affinity chromatography, and cleaved by cyanogen bromide. SDS-PAGE revealed the presence of three proteins with masses of 7 kDa (CT-apoA-I), 18 kDa (apoLp-III), and a minor 26 kDa band of uncleaved chimera. The digest was reloaded on the Ni-affinity column to bind apoLp-III and uncleaved chimera, while CT-apoA-I was washed from the column and collected. Alternatively, CT-apoA-I was isolated from the digest by reversed-phase HPLC. CT-apoA-I was α-helical, highly effective in solubilizing phospholipid vesicles and disaggregating LPS micelles. However, CT-apoA-I was less active compared to full-length apoA-I in protecting lipolyzed low density lipoproteins from aggregating, and disrupting phosphatidylglycerol bilayer vesicles. Thus the novel expression system produced mg quantities of functional CT-apoA-I, facilitating structural and functional studies of this critical domain of apoA-I.

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“…Previous studies have shown that helix 5, in isolation, cannot form complexes with phospholipid in vitro (11). However, quenching of its fluorescence by spin-labeled fatty acids when helix 5 is present in the intact protein indicates that despite its terminal location in the protein sequence, helix 5 contacts the lipid surface in a stable, oriented manner.…”

Section: Fig 2 Effect Of Apolipoproteins On Phospholipase C-inducedmentioning

confidence: 95%

“…Such an organization facilitates its existence as a soluble protein in an aqueous medium, such as plasma, and permits binding to lipid surfaces through a conformational change. To obtain information about lipid-associated conformations of apoLp-III, the fluorescence properties of its unique tyrosine residue (located close to the center of the nonpolar face at position 145 in helix 5) have been investigated (8,10,11,24). In these studies, Tyr-145 served as a reporter of the conformational status of helix 5 in different environments.…”

Section: Fig 2 Effect Of Apolipoproteins On Phospholipase C-inducedmentioning

confidence: 99%

“…Although fluorescence from Tyr-145 (located in helix 5) is negligible in the lipid-free state (10,11), a significant enhancement was observed upon lipid binding (8), which facilitated quenching studies. By comparison of the relative effectiveness of different lipid-based quenchers, information about the spatial alignment of apoLp-III ␣-helices complexed with phospholipids and spherical lipoprotein surfaces was obtained.…”

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confidence: 99%

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Fluorescence Studies of Exchangeable Apolipoprotein-Lipid Interactions

Sahoo

Narayanaswami

Kay

et al. 1998

Journal of Biological Chemistry

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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 ␣-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 K sv values of 27.1 and 19.8 M ؊1 , respectively. Upon binding to the surface of a spherical lipoprotein particle, K sv 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 K sv 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 ␣-helices in phospholipid disc complexes and support the concept that interaction with spherical lipoprotein particles results in superficial contact of apoLp-III helical segments with the monolayer surface, providing a basis for its reversible binding ability.Plasma lipoprotein metabolism is regulated to a large extent by reversible binding of exchangeable apolipoproteins (1, 2). These amphipathic ␣-helical proteins have the ability to exist in both water-soluble and lipid-bound forms, a property which is the basis of their physiological role in lipoprotein metabolism. Apolipophorin III (apoLp-III) 1 from the Sphinx moth, Manduca sexta, is a 166-amino acid member of the exchangeable apolipoprotein family that has been well characterized (3). Structural information is available for the lipid-free form of M. sexta apoLp-III (4), as well as Locusta migratoria apoLp-III (5). Both proteins exist as globular up-and-down amphipathic ␣-helix bundles, with their polar and charged residues facing the aqueous environment and their hydrophobic residues oriented toward the protein interior....

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Structural and Binding Characteristics of the Carboxyl Terminal Fragment of Apolipophorin III from Manduca sexta

Cited by 20 publications

References 51 publications

An N-Terminal Three-Helix Fragment of the Exchangeable Insect Apolipoprotein Apolipophorin III Conserves the Lipid Binding Properties of Wild-Type Protein

An N-Terminal Three-Helix Fragment of the Exchangeable Insect Apolipoprotein Apolipophorin III Conserves the Lipid Binding Properties of Wild-Type Protein

Expression of the C-terminal domain of human apolipoprotein A-I using a chimeric apolipoprotein

Fluorescence Studies of Exchangeable Apolipoprotein-Lipid Interactions

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