An N-terminal three-helix fragment of the exchangeable insect apolipoprotein apolipophorin III conserves the lipid binding properties of wild-type protein

Apolipophorin III (apoLp-III) from the greater wax moth Galleria mellonella is an exchangeable insect apolipoprotein that consists of five amphipathic alpha -helices, sharing high sequence identity with apoLp-III from the sph inx moth Manduca sexta whose structure is available. To define the minimal requirement for apoLp-III structural stability and function, a C-terminal t runcated apoLp-III encompassing residues 1-91 of this 163 amino acid protei n was designed. Far-UV circular dichroism spectroscopy revealed apoLp-III(1 -91) has 50% alpha -helix secondary structure content in buffer (wild-type apoLp-III 86%), increasing to essentially 100% upon interactions with dimyr istoylphosphatidylcholine (DMPC), Guanidine hydrochloride denaturation stud ies revealed similar stability properties for wild-type apoLp-III and apoLp -III(1-91), Resistance to denaturation for both proteins increased substant ially upon association with phospholipid. In the absence of lipid, wild-typ e apoLp-III was monomeric whereas apoLp-III(1 -91) partly formed dimers and trimers. Discoidal apoLp-III(1-91)-DMPC complexes were smaller in diameter (13.5 nm) compared to wild-type apoLp-III (17.7 nm), and more molecules of apoLp-III(1-91) associated with the complexes. Lipid interaction revealed that apoLp-III(1-91) binds to modified spherical lipoprotein surfaces and e fficiently transforms phospholipid vesicles into discoidal complexes. Thus, the first three helices of G. mellonella apoLp-III contain the basic featu res required for maintenance of the structural integrity of the entire prot ein.