SPECTROSCOPIC AND LIPID-BINDING STUDIES ON THE AMINO AND CARBOXYL-TERMINAL FRAGMENTS OF LOCUSTA-MIGRATORIA APOLIPOPHORIN-III

The structural basis for the lipid binding capability of Locusta migra toria apolipophorin III (apoLp-III) was assessed by characterizing the amino and carboxyl terminal halves of the protein. The native molecul e (similar to 20 kDa) was deglycosylated with endoglycosidase F (molec ular mass of deglycosylated species similar to 18 kDa) and cleaved wit h endoproteinase Arg-C to yield two fragments with molecular masses of similar to 9 kDa each. The two fragments were purified by reversed-ph ase HPLC and identified by mass spectrometry, amino acid analysis and N-terminal sequencing as the amino terminal (N9) and carboxyl terminal (C9) halves. Due to the apparent discrepancy of the protease digestio n pattern obtained compared to that expected from the deduced amino se quence of apoLp-III cDNA, we carried out partial amino acid sequencing of the fragments and cDNA sequencing for the entire protein. Circular dichroism spectroscopy of the N9 and C9 peptides revealed that both e xist in buffer in a random coil state. However, addition of-trifluoroe thanol, a helix-inducing agent, resulted in the formation of an alpha- helix, reflecting an innate propensity of the peptides to adopt a heli cal conformation. When cosonicated with dimyristoylphosphatidylcholine (DMPC) both peptides assumed an alpha-helical conformation, indicativ e of interaction with the phospholipid. In the presence of phospholipi ds, a 22 nm blue shift in Trp fluorescence emission was observed in th e case of the C9 peptide, suggesting that the Trp residues are located in a more hydrophobic environment. Electron microscopy revealed that, compared to native apoLp-III, both peptides possessed a reduced abili ty to transform DMPC vesicles to disklike complexes. Further, both the N9 and C9 peptides were unable to interact with lipoprotein surfaces, as evidenced by their inability to prevent turbidity development due to aggregation of human low-density Lipoprotein induced by phospholipa se C. These results show that the isolated amino and carboxyl terminal fragments of the protein, while able to interact with lipids, cannot mimic the functional capacity of the intact protein. Thus, we conclude that, aside from specific amphipathic alpha-helices, structural eleme nts of the intact protein contribute to physiologically relevant lipid binding abilities of apoLp-III.