BINDING OF INSECT APOLIPOPHORIN-III TO DIMYRISTOYLPHOSPHATIDYLCHOLINEVESICLES - EVIDENCE FOR A CONFORMATIONAL CHANGE

Apolipophorin-III (apoLp-III), a hemolymph protein of Manduca sexta, c an reversibly associate with the surface of lipoprotein particles. In order to examine the lipid-associated form of apoLp-III, the present s tudies 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 a verage diameter and width of 18.5 +/- 2.0 nm and 4.8 +/- 0.8 nm, respe ctively, as determined by electron microscopy. ApoLp-III.DMPC complexe s analyzed by pore limiting native gradient PAGE demonstrated that a s ingle 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 eq uilibrium experiments, conducted in an analytical ultracentrifuge, con firmed that only one species of apoLp-III DMPC complex was formed at a n initial lipid to protein molar ratio of 67:1, with an apparent molec ular mass of 642,000. Complexes cross-linked with dimethyl suberimidat e indicate that there are a maximum of 6 apoLp-III molecules per disc. Circular dichroism experiments revealed that apoLp-III becomes essent ially completely a-helical on formation of apoLp-III DMPC complexes. C ompared to apoLp-III in the lipid-free state, apoLp-III DMPC complexes were relatively resistant to denaturation by guanidine HCl, displayin g denaturation transitions with midpoints at 2.2 and 3.7 M guanidine H Cl, respectively. The fluorescence excitation and emission spectra of apoLp-III DMPC complexes demonstrate a large enhancement of tyrosine f luorescence as compared to the lipid-free state, suggesting that a con formational change occurs when apoLp-III associates with a lipid surfa ce. 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. The tyrosine-induced fluorescenc e of the complex was quenched with both Cs+ (K-q = 0.573 M(-1)) and KI (K-q = 0.376 M(-1)). The results presented in this study indicate tha t the conformation of apoLp-III is stabilized when complexed with phos pholipids and suggest that tyrosine fluorescence provides a sensitive method to detect M. sexta apoLp-III interaction with lipid surfaces. B ased on the data obtained, together with the length of ru-helices deri ved from the x-ray crystal structure of Locusta migratoria apoLp-III, we propose a model for the interaction of apoLp-III with phospholipid.