Abstract 263: Isotopic Labeling and Chemical Cross-Linking Reveals Intermolecular Contacts and Dynamics of Apolipoprotein A-I Structure in Reconstituted HDL Particles
Apolipoprotein (apo)A-I has been proposed to adopt a number of different, but related, structures when in contact with lipid. The technique of chemical cross-linking has recently been used to determine the spatial relationships between two molecules of apoA-I in reconstituted high density lipoprotein (rHDL) particles. However, this technique is limited in that it cannot unequivocally distinguish between intramolecular and intermolecular contacts. To address this issue, we have produced two forms of recombinant apoA-I that contain the naturally occurring isotope of nitrogen (N14) or a stable isotope (N15). These forms were mixed 1:1 and then used to produce reconstituted HDL particles with synthetic lipids. The resulting mass shifts (readily detectable in the mass spectrometer) were exploited to unambiguously distinguish between intramolecular (N14 to N14 or N15 to N15) and intermolecular (N14 to N15) cross-linked peptides. An additional benefit of this approach was the ability to identify cross-links with high certainty without the need for peptide fragmentation, allowing for dramatic increases in method sensitivity. We studied highly homogeneous rHDL particles made with the fully saturated phospholipid palmitoyl steroyl phosphatidylcholine (PSPC) to minimize apoA-I conformational dynamics. These particles were 98Å in diameter, contained two molecules of apoA-I, approximately 155 molecules of PC, and were discoidal in shape by cryo EM. We identified 30 cross-links (17 intramolecular, 13 intermolecular) that were overall consistent with the double belt model in which both apoA-I molecules wrap around a bilayer of lipids in an antiparallel orientation. Unambiguous intramolecular linkages between the N- and C-terminal regions of apoA-I were inconsistent with the ‘double super helix’ variant of the belt model. We identified several low abundance cross-links that suggest that the N-terminus may be conformationally dynamic and may spend some time folded back across the molecule as proposed for the ‘belt and buckle’ belt model.
- © 2012 by American Heart Association, Inc.