Abstract 410: 3-Dimensional Conformation of HDL ApoA-I Generated by ABCA1
This report describes the 3-dimensional conformation of nascent HDL apoA-I particles generated via ATP binding cassette transporter A1 (ABCA1). Three main types of particles were consistently obtained from; overexpressing HEK293 cells, bone marrow derived mouse macrophages and HepG2 cells. Particles were separated by FPLC and sized using gel electrophoresis and electron microscopy. Total lipid compositions were obtained from HPLC and mass spectrometry and protein conformation by chemical cross-linking and mass spectrometry (CCLMS). The first particle type corresponded to a lipid-rich nascent HDL (nHDL) between 13-10 nm in diameter, containing 3 molecules of apoA-I, with 45% of their total lipid as cholesterol, 35% as phosphatidylcholine and 20% as sphingomyelin with a total of ∼250 molecules of lipid per particle. The second type corresponded to 7-8 nm diameter particle containing 2 molecules of apoA-I and 26 total lipid molecules, while the third particle type was a monomer of apoA-I with 3 lipid molecules. Three-dimensional information was derived from the 13-10 nm nHDL particles by first crosslinking these purified particles and separating the monomeric, dimeric and trimeric apoA-I bands by SDS-PAGE. These CCL products were subjected to in-gel trypsin digest and LC-MS/MS. Reconstructed MS total-ion chromatograms were evaluated for CCL peptides with identity verified by MS/MS sequencing. Using several lysine-specific crosslinkers with arm lengths from 12Å to 36Å we compiled the distribution of CCLs. The same analysis was performed for the 7-8 nm nHDL that only yielded dimmer and monomer. Several intramolecular, interpeptide and intermolecular crosslinks were identified for each type category of nHDL. CCLs from the 7-8 nm nHDL suggested that the 2 apoA-I molecules assumed antiparallel counter-rotating helices. Tighter bending of the double belt was required to cover the 26 lipid molecules. Crosslinking of the 13-10 nm nHDL suggested that all three molecules were antiparallel to one another. The conformation that fit the CCLs was a belt with the middle apoA-I molecule antiparallel to the two outer apoA-I molecules. Overall, these data suggest that apoA-I adapts its conformation to encompass available lipid, but with larger amounts of lipid a belt conformation is preferred.
- © 2012 by American Heart Association, Inc.