Binding of insect apolipophorin III to dimyristoylphosphatidylcholine vesicles. Evidence for a conformational change

Apolipophorin-III (apoLp-III), a hemolymph protein of Manduca sexta, can reversibly associate with the surface of lipoprotein particles. In order to examine the lipid-associated form of apoLp-III, the present studies investigate the structure and properties of apoLp-III complexes with dimyristoylpho...

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Veröffentlicht in:The Journal of biological chemistry 1994-02, Vol.269 (6), p.4605-4612
Hauptverfasser: Wientzek, M, Kay, C.M, Oikawa, K, Ryan, R.O
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container_title The Journal of biological chemistry
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creator Wientzek, M
Kay, C.M
Oikawa, K
Ryan, R.O
description Apolipophorin-III (apoLp-III), a hemolymph protein of Manduca sexta, can reversibly associate with the surface of lipoprotein particles. In order to examine the lipid-associated form of apoLp-III, the present studies investigate the structure and properties of apoLp-III complexes with dimyristoylphosphatidylcholine (DMPC). Association of apoLp-III with DMPC vesicles results in the formation of uniform discs with an average diameter and width of 18.5 +/- 2.0 nm and 4.8 +/- 0.8 nm, respectively, as determined by electron microscopy. ApoLp-III-DMPC complexes analyzed by pore-limiting native gradient PAGE demonstrated that a single major species of complex was formed within a wide range of lipid to protein molar ratios (DMPC:apoLP-III; 13:1 to 360:1). Flotation equilibrium experiments, conducted in an analytical ultracentrifuge, confirmed that only one species of ApoLp-III-DMPC complex was formed at an initial lipid to protein molar ratio of 67:1, with an apparent molecular mass of 642,000. Complexes cross-linked with dimethyl suberimidate indicate that there are a maximum of 6 apoLp-III molecules per disc. Circular dichroism experiments revealed that apoLp-III becomes essentially completely alpha-helical on formation of apoLp-III-DMPC complexes. Compared to apoLp-III in the lipid-free state, apoLp-III-DMPC complexes were relatively resistant to denaturation by guanidine HCl, displaying denaturation transitions with midpoints at 2.2 and 3.7 m guanidine HCl, respectively. The fluorescence excitation and emission spectra of ApoLp-III-DMPC complexes demonstrate a large enhancement of tyrosine fluorescence as compared to the lipid-free state, suggesting that a conformational change occurs when apoLp-III associates with a lipid surface. Denaturation of apoLp-III in the complex by guanidine HCl resulted in a tyrosine fluorescence level similar to that of lipid-free apoLp-III in the presence of guanidine HCl
doi_str_mv 10.1016/S0021-9258(17)41819-9
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Evidence for a conformational change</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>Apolipophorin-III (apoLp-III), a hemolymph protein of Manduca sexta, can reversibly associate with the surface of lipoprotein particles. In order to examine the lipid-associated form of apoLp-III, the present studies investigate the structure and properties of apoLp-III complexes with dimyristoylphosphatidylcholine (DMPC). Association of apoLp-III with DMPC vesicles results in the formation of uniform discs with an average diameter and width of 18.5 +/- 2.0 nm and 4.8 +/- 0.8 nm, respectively, as determined by electron microscopy. ApoLp-III-DMPC complexes analyzed by pore-limiting native gradient PAGE demonstrated that a single major species of complex was formed within a wide range of lipid to protein molar ratios (DMPC:apoLP-III; 13:1 to 360:1). Flotation equilibrium experiments, conducted in an analytical ultracentrifuge, confirmed that only one species of ApoLp-III-DMPC complex was formed at an initial lipid to protein molar ratio of 67:1, with an apparent molecular mass of 642,000. Complexes cross-linked with dimethyl suberimidate indicate that there are a maximum of 6 apoLp-III molecules per disc. Circular dichroism experiments revealed that apoLp-III becomes essentially completely alpha-helical on formation of apoLp-III-DMPC complexes. Compared to apoLp-III in the lipid-free state, apoLp-III-DMPC complexes were relatively resistant to denaturation by guanidine HCl, displaying denaturation transitions with midpoints at 2.2 and 3.7 m guanidine HCl, respectively. The fluorescence excitation and emission spectra of ApoLp-III-DMPC complexes demonstrate a large enhancement of tyrosine fluorescence as compared to the lipid-free state, suggesting that a conformational change occurs when apoLp-III associates with a lipid surface. 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Evidence for a conformational change</title><author>Wientzek, M ; Kay, C.M ; Oikawa, K ; Ryan, R.O</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c457t-b6d984db2750f4ee574ea46ef24f76d066d307db6d6ae1c1563a4b07049286e23</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1994</creationdate><topic>Analytical, structural and metabolic biochemistry</topic><topic>Animals</topic><topic>Apolipoproteins - chemistry</topic><topic>Apolipoproteins - metabolism</topic><topic>Biological and medical sciences</topic><topic>Dimyristoylphosphatidylcholine - chemistry</topic><topic>Dimyristoylphosphatidylcholine - metabolism</topic><topic>Fundamental and applied biological sciences. 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Evidence for a conformational change</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>1994-02-11</date><risdate>1994</risdate><volume>269</volume><issue>6</issue><spage>4605</spage><epage>4612</epage><pages>4605-4612</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><coden>JBCHA3</coden><abstract>Apolipophorin-III (apoLp-III), a hemolymph protein of Manduca sexta, can reversibly associate with the surface of lipoprotein particles. In order to examine the lipid-associated form of apoLp-III, the present studies investigate the structure and properties of apoLp-III complexes with dimyristoylphosphatidylcholine (DMPC). Association of apoLp-III with DMPC vesicles results in the formation of uniform discs with an average diameter and width of 18.5 +/- 2.0 nm and 4.8 +/- 0.8 nm, respectively, as determined by electron microscopy. ApoLp-III-DMPC complexes analyzed by pore-limiting native gradient PAGE demonstrated that a single major species of complex was formed within a wide range of lipid to protein molar ratios (DMPC:apoLP-III; 13:1 to 360:1). Flotation equilibrium experiments, conducted in an analytical ultracentrifuge, confirmed that only one species of ApoLp-III-DMPC complex was formed at an initial lipid to protein molar ratio of 67:1, with an apparent molecular mass of 642,000. Complexes cross-linked with dimethyl suberimidate indicate that there are a maximum of 6 apoLp-III molecules per disc. Circular dichroism experiments revealed that apoLp-III becomes essentially completely alpha-helical on formation of apoLp-III-DMPC complexes. Compared to apoLp-III in the lipid-free state, apoLp-III-DMPC complexes were relatively resistant to denaturation by guanidine HCl, displaying denaturation transitions with midpoints at 2.2 and 3.7 m guanidine HCl, respectively. The fluorescence excitation and emission spectra of ApoLp-III-DMPC complexes demonstrate a large enhancement of tyrosine fluorescence as compared to the lipid-free state, suggesting that a conformational change occurs when apoLp-III associates with a lipid surface. Denaturation of apoLp-III in the complex by guanidine HCl resulted in a tyrosine fluorescence level similar to that of lipid-free apoLp-III in the presence of guanidine HCl</abstract><cop>Bethesda, MD</cop><pub>American Society for Biochemistry and Molecular Biology</pub><pmid>8308032</pmid><doi>10.1016/S0021-9258(17)41819-9</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record>
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subjects Analytical, structural and metabolic biochemistry
Animals
Apolipoproteins - chemistry
Apolipoproteins - metabolism
Biological and medical sciences
Dimyristoylphosphatidylcholine - chemistry
Dimyristoylphosphatidylcholine - metabolism
Fundamental and applied biological sciences. Psychology
HEMOLINFA
HEMOLYMPHE
In Vitro Techniques
LECITHINE
LECITINAS
Lepidoptera
Lipid Bilayers
LIPOPROTEINAS
LIPOPROTEINE
Lipoproteins, myelin
MANDUCA SEXTA
Membrane Lipids - metabolism
Microscopy, Electron
Molecular Weight
Moths
Protein Binding
Protein Conformation
Protein Denaturation
Protein Structure, Secondary
PROTEINAS
PROTEINE
Proteins
Spectrometry, Fluorescence
Sphingidae
title Binding of insect apolipophorin III to dimyristoylphosphatidylcholine vesicles. Evidence for a conformational change
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