title: Conformational adaptation of apolipoprotein A-I to discretely sized phospholipid complexes.
abstract: The conformational constraints for apoA-I bound to recombinant phospholipid complexes (rHDL) were attained from a combination of chemical cross-linking and mass spectrometry. Molecular distances were then used to refine models of lipid-bound apoA-I on both 80 and 96 A diameter rHDL particles. To obtain molecular constraints on the protein bound to phospholipid complexes, three different lysine-selective homo-bifunctional cross-linkers with increasing spacer arm lengths (i.e., 7.7, 12.0, and 16.1 A) were reacted with purified, homogeneous recombinant 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) apoA-I rHDL complexes of each diameter. Cross-linked dimeric apoA-I products were separated from monomeric apoprotein using 12% SDS-PAGE, then subjected to in-gel trypsin digest, and identified by MS/MS sequencing. These studies aid in the refinement of our previously published molecular model of two apoA-I molecules bound to approximately 150 molecules of POPC and suggest that the protein hydrophobic interactions at the N- and C-terminal domains decrease as the number of phospholipid molecules or "lipidation state" of apoA-I increases. Thus, it appears that these incremental changes in the interaction between the N- and C-terminal ends of apoA-I stabilize its tertiary conformation in the lipid-free state as well as allowing it to unfold and sequester discrete amounts of phospholipid molecules.
authors.affiliation: Departments of Pathology, Center for Lipid Science, Wake Forest University Medical Center, 391 Technology Way, Building A1, Winston-Salem, North Carolina 27101, USA.
authors.forename: Shaila
authors.initials: S
authors.lastname: Bhat
authors.forename: Mary G
authors.initials: MG
authors.lastname: Sorci-Thomas
authors.forename: Rubina
authors.initials: R
authors.lastname: Tuladhar
authors.forename: Michael P
authors.initials: MP
authors.lastname: Samuel
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Thomas
mesh_terms: D016632:Apolipoprotein A-I / Q000737:chemistry*; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008075:Lipoproteins, HDL / Q000737:chemistry*; D008958:Models, Molecular; D010713:Phosphatidylcholines / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013388:Succinimides / Q000737:chemistry; D053719:Tandem Mass Spectrometry
chemical_list: D016632:Apolipoprotein A-I; D003432:Cross-Linking Reagents; D008075:Lipoproteins, HDL; D010713:Phosphatidylcholines; D010743:Phospholipids; D011994:Recombinant Proteins; D013388:Succinimides; C096464:disuccinimidyl glutarate; C028722:ethylene glycolylbis(succinimidyl succinate); C011240:dithiobis(succinimidylpropionate); C028694:1-palmitoyl-2-oleoylphosphatidylcholine