Factors Affecting the Stability and Conformation of Locusta migratoria Apolipophorin III

Pm, Weers; Cm, Kay; Oikawa, Kimio; Wientzek, Monika; Dj, Van der Horst; Ryan, RO

doi:10.1021/bi00178a019

Cited by 59 publications

(70 citation statements)

References 32 publications

(46 reference statements)

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“…Consistent with the known lability of exchangeable amphipathic apolipoproteins (Edelstein and Scanu, 1980;Weinberg and Spector, 1985), the observed transition midpoints of reduced and oxidized N40C/L90C apoLp-III were 0.25 and 0.43 M GdnHCl, respectively. These values are comparable with those of 0.36 M for WT M. sexta apoLp-III and 0.6 M for L. migratoria apoLp-III (Weers et al, 1994) and suggest that no major structural perturbations in N40C/L90C apoLp-III resulted from the mutations and disulfide bond formation.…”

Section: Evaluation Of Disulfide Bond Formation and Structural Characsupporting

confidence: 72%

Disulfide Bond Engineering to Monitor Conformational Opening of Apolipophorin III During Lipid Binding

Narayanaswami¹,

Wang²,

Kay

et al. 1996

Journal of Biological Chemistry

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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 disclike 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.Exchangeable apolipoproteins belong to a class of amphipathic ␣-helical proteins that regulate the metabolism and dynamics of lipoprotein interconversions. These proteins reversibly associate with the surface of lipoprotein particles in response to hydrophobic surface availability or their intrinsic ability to displace pre-existing apolipoproteins. This functional property implies an ability to exist in both lipid-free and lipidbound forms in plasma, and it has been proposed that a dramatic conformational change is required for initiation and maintenance of interaction with lipid surfaces (Breiter et al., 1991;Weisgraber, 1994). The sole exchangeable apolipoprotein found in insect hemolymph, apolipophorin III (apoLp-III), 1 provides an excellent model to study lipid association-induced conformational changes of amphipathic exchangeable apolipoproteins. ApoLp-III is well characterized in terms of physicochemical and functional properties (see Van der Horst, 1990;Blacklock and Ryan, 1994; Soulages and Wells, 1994, for reviews). Structural informa...

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Section: Evaluation Of Disulfide Bond Formation and Structural Characsupporting

confidence: 72%

Disulfide Bond Engineering to Monitor Conformational Opening of Apolipophorin III During Lipid Binding

Narayanaswami¹,

Wang²,

Kay

et al. 1996

Journal of Biological Chemistry

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“…In this model, interaction with a lipid surface induces helixbundle opening about hinge loops at one end of the molecule, thereby exposing a continuous hydrophobic surface capable of binding to lipid surfaces. This hypothesis is widely accepted because it is supported by considerable experimental evidence, obtained by using different approaches (9)(10)(11)(12)(13)(14). Using NMR techniques, evidence for a conformational adaptation of apoLp-III was obtained at the amino acid level (24).…”

Section: Resultsmentioning

confidence: 68%

Structural basis for the conformational adaptability of apolipophorin III, a helix-bundle exchangeable apolipoprotein

Wang

Sykes

Ryan³

2002

Proc. Natl. Acad. Sci. U.S.A.

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120

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The high-resolution NMR structure of apolipophorin III from the sphinx moth, Manduca sexta, has been determined in the lipid-free state. We show that lipid-free apolipophorin III adopts a unique helix-bundle topology that has several characteristic structural features. These include a marginally stable, up-and-down helix bundle that allows for concerted opening of the bundle about ''hinged'' loops upon lipid interaction and buried polar͞ionizable residues and buried interhelical H-bonds located in the otherwise hydrophobic interior of the bundle that adjust protein stability and facilitate lipid-induced conformational opening. We suggest that these structural features modulate the conformational adaptability of the lipid-free helix bundle upon lipid binding and control return of the open conformation to the original lipid-free helixbundle state. Taken together, these data provide a structural rationale for the ability of exchangeable apolipoproteins to reversibly interact with circulating lipoprotein particles. E xchangeable apolipoproteins constitute a functionally important family of proteins that play critical roles in lipid transport and lipoprotein metabolism (1). Available structural and biophysical data on members of this protein family indicate that lipid-free exchangeable apolipoproteins share a common molecular architecture, the elongated up-and-down amphipathic ␣-helix bundle (2-4). Contact with a lipid surface induces a conformational change in the helix bundle, wherein the protein opens about ''hinged'' loops that connect helical segments (3,5). The open, lipid-associated conformation appears to be an extended ␣-helical structure that presents a continuous hydrophobic surface. A recently published NMR structure of apolipoprotein C-I in a lipid-mimetic environment supports this model (6). Further, the x-ray crystal structure of a human apolipoprotein A-I (apoA-I) N-terminal truncation mutant, apoA-I(⌬1-43), shows a continuously curved, extended ␣-helical structure that represents a putative lipid-bound conformation of this protein (7). A recently published secondary structure of an apoA-I C-terminal truncation mutant, apoA-I(⌬187-243), in the presence of SDS micelles, indicates a similar location of helix segments as shown in the x-ray crystal structure, although no tertiary interactions were observed between helices (8). Other experimental data, including monolayer balance (9), fluorescence spectroscopy (10), near UV circular dichroism spectroscopy (11), surface plasmon resonance spectroscopy (12), Fourier-transformed IR spectroscopy (13), and disulfide-bond engineering (14) support a conformational opening in helixbundle exchangeable apolipoproteins upon lipid binding. This conformational adaptability raises the question as to which structural features in the lipid-free helix bundle regulate this process, and this question remains unanswered.Apolipophorin III (apoLp-III) is a prototypical exchangeable apolipoprotein found in many insect species that functions in transport of diacylglycerol (DAG) fr...

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“…A typical 50-ml culture yielded 4 mg of protein with a specific activity of 6 ϫ 10 6 dpm/mg. CD Spectroscopy-A Jasco J-720 spectropolarimeter, interfaced to an Epson Equity 386/25 computer controlled by Jasco software, was used to analyze the ␣-helical content of mutant and wild-type (WT) apoLpIIIs and to monitor temperature-or guanidine hydrochloride-induced denaturation (8). A modified Contin program by Provencher and Glöck-ner (20), which contains poly-L-glutamate as a helical reference standard, was used to estimate the ␣-helical content of the proteins.…”

Section: Methodsmentioning

confidence: 99%

“…The availability of (i) high resolution structural information (7), (ii) biophysical data (8,9), and (iii) a bacterial system for expression of recombinant apoLp-III (10) establishes this as an excellent model system to study structure-function relationships of amphipathic exchangeable apolipoproteins. X-ray crystallography data showed that the lipid-free form of apoLp-III from Locusta migratoria is composed of a bundle of five amphipathic antiparallel ␣-helices (7).…”

mentioning

confidence: 99%

Interaction of an Exchangeable Apolipoprotein with Phospholipid Vesicles and Lipoprotein Particles

Weers¹,

Narayanaswami²,

Kay³

et al. 1999

Journal of Biological Chemistry

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Factors Affecting the Stability and Conformation of Locusta migratoria Apolipophorin III

Cited by 59 publications

References 32 publications

Disulfide Bond Engineering to Monitor Conformational Opening of Apolipophorin III During Lipid Binding

Disulfide Bond Engineering to Monitor Conformational Opening of Apolipophorin III During Lipid Binding

Structural basis for the conformational adaptability of apolipophorin III, a helix-bundle exchangeable apolipoprotein

Interaction of an Exchangeable Apolipoprotein with Phospholipid Vesicles and Lipoprotein Particles

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