title: Combined N- and C-terminal truncation of human apolipoprotein A-I yields a folded, functional central domain.
abstract: A combined N- and C-terminal truncation variant of human apolipoprotein A-I (apoA-I) was designed, expressed in Escherichia coli, isolated, and characterized. Hydrodynamic experiments yielded a weight average molecular weight of 34000, indicating apoA-I-(44-186) exists in solution predominantly as a dimer. An axial ratio of 4.2 was calculated for the dimer based on sedimentation velocity experiments. Far-UV circular dichroism spectroscopy of apoA-I-(44-186) in buffer indicated the presence of 65% alpha-helix secondary structure. Guanidine hydrochloride denaturation experiments yielded a transition midpoint of 0.5 M for apoA-I-(44-186). ApoA-I-(44-186) induced solubilization of dimyristoylphosphatidylcholine vesicles at a rate comparable to that of full-length apoA-I, displayed lipoprotein binding ability, and was an acceptor of ABCA1-mediated cholesterol efflux from cultured macrophages. Fluorescence quenching studies with KI indicate that the three Trp residues in apoA-I-(44-186) are shielded from the aqueous environment. Taken together, the data indicate that lipid-free apoA-I-(44-186) adopts a folded conformation in solution that possesses lipid binding capability. The central region of apoA-I appears to adopt a globular amphipathic alpha-helix bundle organization that is stabilized by intramolecular and/or intermolecular helix-helix interactions. Lipid association likely results in a conformational adaptation wherein helix-helix contacts are substituted for helix-lipid interactions.
authors.affiliation: Lipid Biology in Health and Disease Research Group, Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, California 94609, USA.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Beckstead
authors.forename: Brian L
authors.initials: BL
authors.lastname: Block
authors.forename: John K
authors.initials: JK
authors.lastname: Bielicki
authors.forename: Cyril M
authors.initials: CM
authors.lastname: Kay
authors.forename: Michael N
authors.initials: MN
authors.lastname: Oda
authors.forename: Robert O
authors.initials: RO
authors.lastname: Ryan
mesh_terms: D000818:Animals; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002942:Circular Dichroism; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D008027:Light; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D012542:Scattering, Radiation; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence
chemical_list: D016632:Apolipoprotein A-I; D010446:Peptide Fragments; D004134:Dimyristoylphosphatidylcholine