Evidence for Lipid-Dependent Structural Changes in Specific Domains of Apolipoprotein B100^†

The structural organization and stability of apoB100 in complexes containing triglyceride (TG) and phospholipid have been examined. LDL was delipidated to form aqueous soluble apoB100−TG complexes that retain 70% of LDL TG, but contain no other lipids. The apoB100−TG complexes exhibited reduced amphipathic α-helical content (17%) and net negative charge (−2.9 mV) as compared to native LDL−apoB100 (49% and −6 mV, respectively). Of 28 anti-apoB monoclonal antibodies tested, 15 showed partial or full reactivity with apoB100−TG. The immunoreactive epitopes of apoB100−TG were restricted to those situated in either the amino terminal globular domain (4 of 6) or in regions of apoB100 that are predicted to be composed of amphipathic β-strands (11 of 13). Incubation of the apoB100−TG complex with palmitoyloleoylphosphatidylcholine (POPC) spontaneously (<10 min) formed homogeneous lipoproteins (20 nm) that contained approximately 300 molecules of POPC per particle (apoB100−PC). Phospholipidation of apoB100−TG complexes partially recovered the α-helical content (34%) and net negative charge (−4.9 mV) of the native LDL and restored resistance of apoB100 to denaturation by guanidine HCl (5.8 M). Addition of phospholipids to apoB100−TG also increased the immunoreactivity of specific epitopes that are located primarily in regions of apoB100 that are thought to be constituted of amphipathic β-strands. The effects of TG and phospholipid on apoB100 conformation appear to be highly domain-specific. On the basis of these results, we propose that the β-strands of apoB100 may represent a nonflexible lipid-associating backbone, while the amphipathic α-helical domains may represent flexible lipid-binding regions that allow the particle to accommodate varying amounts of lipid.