Abstract 223: The Structural Organization of Apolipoprotein A-I in Reconstituted High-Density Lipoprotein Discs Using Stable Isotope-Assisted Cross-Linking
X-ray crystallography studies of lipid-free apolipoprotein A-I (apoA-I) and related apoA-IV have shown that the proteins form a reciprocating dimer which could be directly related to the structural organization of lipid-bound apoA-I. Previous work has demonstrated the success of chemical cross-linking combined with mass spectrometry to derive distance constraints for molecular modeling of apoA-I in reconstituted high density lipoprotein (HDL) particles. However, these studies have been limited by i) the relative paucity of distance constraint data, and ii) ambiguities in determining the molecular span of the cross-links. Here, we have addressed the first issue by using an affinity-tagged cross-linker to purify cross-linked peptides away from non-modified peptides prior to sampling with mass spectrometry. This essentially boosts the signal from cross-linked peptides of interest that would have otherwise been suppressed by detection of non-modified peptides. Using this method, we identified >25 cross-linked peptides; the most reported to date for these particles. To address the issue of molecular span, we used a stable isotopic-labeling approach in which wild-type human recombinant apoA-I (14N) was mixed 1:1 with an isotopically labeled (15N) apoA-I. Analysis of the isotopic patterns of the resulting mass spectra allowed the unambiguous determination of the molecular span of the cross-linked peptides; i.e. whether the links were within the same molecule of apoA-I (intramolecular) or between the dimers of apoA-I (intermolecular). This information will be used to derive a detailed novel model of lipid-bound apoA-I in reconstituted HDL, an important step in our overall understanding of how native HDL species are organized and how they interact with plasma remodeling factors and cell surface proteins.
Author Disclosures: J. Melchior: None. J. Morris: None. W.S. Davidson: None.
- © 2014 by American Heart Association, Inc.