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...
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Published in | The Journal of biological chemistry Vol. 271; no. 43; pp. 26855 - 26862 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Society for Biochemistry and Molecular Biology
25.10.1996
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Subjects | |
Online Access | Get full text |
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Summary: | 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. |
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Bibliography: | ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 |
ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.271.43.26855 |