Fluorescence Spectroscopy of Single Tryptophan Mutants of Apolipophorin-III in Discoidal Lipoproteins of Dimyristoylphosphatidylcholine

Jl, Soulages; El, Arrese

doi:10.1021/bi0007223

Cited by 22 publications

(16 citation statements)

References 27 publications

(49 reference statements)

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“…Thus, a fully extended polypeptide chain would produce essentially the same results that were observed in this study. In fact, on the basis of the results of this study as well as on the conclusions of recent studies reporting on the topology and lipid-interacting regions of the apolipoprotein (17,18), a structural model of apoLp-III in discoidal lipoproteins is proposed below. In this model it is assumed that the apolipoprotein adopts a fully extended helical conformation.…”

Section: Conformation Of Apolp-iii In the Lipid-bound State Inferred mentioning

confidence: 76%

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Structure of Apolipophorin-III in Discoidal Lipoproteins

Garda

Arrese

Soulages

2002

Journal of Biological Chemistry

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The structure of apolipophorin III in the lipid-bound state and the extent of the conformational change that takes place when the five-helix bundle apolipoprotein binds to a lipoprotein lipid surface were investigated by fluorescence resonance energy transfer in discoidal lipoproteins. Four intramolecular interhelical distances between helix pairs 1-4, 2-4, 3-4, and 5-4 were estimated by fluorescence resonance energy transfer in both the lipid-free and the lipid-bound states. Depending on the helices pairs, the intramolecular interhelical distances increased between 15 and > 20 Å upon binding of the apolipoprotein to lipid, demonstrating for the first time that binding to lipid is accompanied by a major change in interhelical distances. Using discoidal lipoproteins made with a combination of apolipophorin III molecules containing donor and acceptor groups and apolipophorin III molecules containing neither donor nor acceptor groups, it was possible to obtain information about intermolecular interhelical distances between the helix 4 of one apolipoprotein and the helices 1, 2, 3, and 5 of a second apolipoprotein residing in the same discoidal lipoprotein. Altogether, the estimated intermolecular and intramolecular interhelical distances suggest a model in which the apolipoprotein arranges in pairs of antiparallel and fully extended polypeptide chains surrounding the periphery of the bilayer disc.Exchangeable apolipoproteins represent a group of proteins characterized by their ability to reversibly bind to lipoprotein lipid surfaces (1). These apolipoproteins play a key role in the regulation of lipid metabolism (2). The interaction of lipoproteins with receptors (3-4) and the exchange of lipids between lipoproteins and lipoproteins and cells (5-7) are among the processes regulated by the structure of apolipoproteins in both the lipid-free and the lipid-bound states. A full understanding of the function of exchangeable apolipoproteins in the homeostasis of lipid metabolism and associated pathologies requires determining the structure of the protein in the lipid-bound state.Apolipophorin-III is a 17-kDa exchangeable apolipoprotein that is involved in the transport of lipids in the hemolymph of insects (8 -9). The structure of apoLp-III 1 in the lipid-free state was solved by x-ray crystallography in 1991 (10). ApoLp-III is composed of a bundle of five amphipathic ␣-helices connected by short loops (10). The study of the structure-function relationship of Locusta migratoria apoLp-III is of interest because the knowledge of the crystal structure of the full-length apoLp-III molecule offers the possibility of relating the structure of an apolipoprotein to its function. Because apoLp-III shares many physical-chemical properties with exchangeable apolipoproteins of humans and other vertebrates (1,(11)(12), it is likely that elucidation of the mechanism of interaction of apoLp-III with lipids would be useful to the understanding of the function of human apolipoproteins.Solving the structure of apolipoproteins in the lipid...

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Section: Conformation Of Apolp-iii In the Lipid-bound State Inferred mentioning

confidence: 76%

“…The first of these studies, which used water-soluble quenchers, suggested that at least three of the five ␣-helices (helices 1, 4, and 5) interact with the lipid surface (17). The second study, which used lipid quenchers, suggested that in discoidal lipoproteins all ␣-helices of apoLp-III interact with the phospholipid acyl chains (18).…”

Section: Discussionmentioning

confidence: 99%

Structure of Apolipophorin-III in Discoidal Lipoproteins

Garda

Arrese

Soulages

2002

Journal of Biological Chemistry

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“…Protein Expression and Purification-Thioredoxin-apoLp-III fusion proteins were overexpressed and isolated from the bacteria as previously reported (13,14). The fusion proteins were purified by standard nickel affinity chromatography.…”

Section: Methodsmentioning

confidence: 99%

Essential Role of the Conformational Flexibility of Helices 1 and 5 on the Lipid Binding Activity of Apolipophorin-III

Soulages

Arrese

Chetty

et al. 2001

Journal of Biological Chemistry

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by Cys residues provided a protein that under nonreducing conditions was fully oxidized. The far-UV CD spectra of this mutant in the reduced and oxidized states indicated that their secondary structures were identical to the structure of the wild type recombinant apoLp-III, which contains no Cys residues. Near-UV CD studies confirmed the formation of a disulfide bond and the absence of structural perturbations. The lipid binding activity of the reduced mutant, as determined by its ability to form discoidal lipoproteins, was nearly identical to that of the wild type protein. Contrarily, the disulfide form of the mutant was not able to form discoidal lipoproteins with liposomes of either dimirystoylphosphatidylcholine or dimyristoylphosphatidylglycerol. It is concluded that the separation of the helices 1 and 5 constitutes one of the key steps along the complex pathway for the formation of the final apolipoprotein lipid-bound state. It is inferred that the conformational flexibility of helices 1 and 5 is a key property of apoLp-III, allowing the exposure of hydrophobic protein regions and the interaction of the hydrophobic faces of the amphipathic ␣-helices with the lipoprotein lipid surface. Apolipophorin-III (apoLp-III)1 is an exchangeable apolipoprotein found in the hemolymph of many insect species in a lipid-free state or bound to the major insect lipoprotein, lipophorin. (1, 2). The structure of apoLp-III from two different insect species has been solved. The structures of Locusta migratoria and Manduca sexta apoLp-III were determined by x-ray crystallography (3) and NMR spectroscopy (4), respectively. The structure of apoLp-III is described as an elongated bundle of five amphipathic ␣-helices, where the nonpolar faces of the helices are oriented toward the protein core. A similar structure, but consisting of a four-helix bundle, is present in the N-terminal domain of human apoE (5). M. sexta and L. migratoria apoLp-III share a large number of physical-chemical properties with the apolipoproteins from humans and other vertebrates (1, 6, 7). Among the common features of exchangeable apolipoproteins are their ability to reversibly bind to lipoprotein surfaces, their content and type of amphipathic ␣-helices (6), and their ability to form discoidal lipoprotein particles (8).The association of apolipoproteins with lipids is a complex process that involves several steps including protein conformational changes and the disruption of the structure of the phospholipid monolayer or bilayer. Because of this complexity, the elucidation of the limiting steps and energy barriers involved in the formation of lipoproteins is also a complex task. Two processes were clearly distinguished in binding of apoLp-III to a planar lipid bilayer (9); the first process consisted in an adsorption of apoLp-III to the phospholipid bilayer, whereas the second process involved the insertion of the protein into the lipid bilayer and the formation of lipoprotein complexes (9). A recent study with disulfide mutants of apoE has shown that three...

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“…Steady-state fluorescence studies showed a strong change in the properties of tryptophan (L. migratoria) or tyrosine (M. sexta) upon binding to DMPC, indicative of their transition to a lipid environment Narayanaswami et al, 1996a;Weers et al, 2000b). This transition was observed for each helix (Soulages and Arrese, 2000). Near and Far UV CD analysis showed dramatic changes in spectra upon lipid binding, indicative of a large conformational change (Weers et al, 1994).…”

Section: Lipid-bound Conformationmentioning

confidence: 99%