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 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