title: Structural determination of lipid-bound ApoA-I using fluorescence resonance energy transfer.
abstract: Based on the x-ray crystal structure of lipid-free Delta43 apoA-I, two monomers of apoA-I were suggested to bind to a phospholipid bilayer in an antiparallel paired dimer, or "belt orientation." This hypothesis challenges the currently held model in which each of the two apoA-I monomers fold as antiparallel alpha-helices or "picket fence orientation." When apoA-I is bound to a phospholipid disc, the first model predicts that the glutamine at position 132 on one apoA-I molecule lies within 16 A of glutamine 132 in the second monomer, whereas, the second model predicts glutamines at position 132 to be 104 A apart. To distinguish between these models, glutamine at position 132 was mutated to cysteine in wild-type apoA-I to produce Q132C apoA-I, which were labeled with thiol-reactive fluorescent probes. Q132C apoA-I was labeled with either fluorescein (donor probe) or tetramethylrhodamine (acceptor probe) and then used to make recombinant phospholipid discs (recombinant high density lipoprotein (rHDL)). The rHDL containing donor- and acceptor-labeled Q132C apoA-I were of similar size, composition, and lecithin:cholesterol acyltransferase reactivity when compared to rHDL-containing human plasma apoA-I. Analysis of donor probe fluorescence showed highly efficient quenching in rHDL containing one donor- and one acceptor-labeled Q132C apoA-I. rHDL containing only acceptor probe-labeled Q132C apoA-I showed rhodamine self-quenching. Both of these observations demonstrate that position 132 in two lipid-bound apoA-I monomers were in close proximity, supporting the "belt conformation" hypothesis for apoA-I on rHDL.
authors.affiliation: Departments of Pathology, Microbiology and Immunology, and Biochemistry, The Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA.
authors.forename: H
authors.initials: H
authors.lastname: Li
authors.forename: D S
authors.initials: DS
authors.lastname: Lyles
authors.forename: M J
authors.initials: MJ
authors.lastname: Thomas
authors.forename: W
authors.initials: W
authors.lastname: Pan
authors.forename: M G
authors.initials: MG
authors.lastname: Sorci-Thomas
mesh_terms: D016632:Apolipoprotein A-I / Q000737:chemistry*; D018360:Crystallography, X-Ray; D005454:Fluorescence Polarization; D006801:Humans; D008055:Lipids; D011487:Protein Conformation; D013329:Structure-Activity Relationship
chemical_list: D016632:Apolipoprotein A-I; D008055:Lipids