title: The carboxyl-terminal segment of apolipoprotein A-V undergoes a lipid-induced conformational change.
abstract: Apolipoprotein (apo) A-V is a 343-residue, multidomain protein that plays an important role in regulation of plasma triglyceride homeostasis. Primary sequence analysis revealed a unique tetraproline sequence (Pro293-Pro296) near the carboxyl terminus of the protein. A peptide corresponding to the 48-residue segment beyond the tetraproline motif was generated from a recombinant apoA-V precursor wherein Pro295 was replaced by Met. Cyanogen bromide cleavage of the precursor protein, followed by negative affinity chromatography, yielded a purified peptide. Nondenaturing polyacrylamide gel electrophoresis verified that apoA-V(296-343) solubilizes phospholipid vesicles, forming a relatively heterogeneous population of reconstituted high-density lipoprotein with Stokes' diameters >17 nm. At the same time, apoA-V(296-343) failed to bind a spherical lipoprotein substrate in vitro. Far-UV circular dichroism spectroscopy revealed the peptide is unstructured in buffer yet adopts significant alpha-helical secondary structure in the presence of the lipid mimetic solvent trifluoroethanol (TFE; 50% v/v). Heteronuclear multidemensional NMR spectroscopy experiments were conducted with uniformly (15)N- and (15)N/(13)C-labeled peptide in 50% TFE. Peptide backbone assignment and secondary structure prediction using TALOS+ reveal the peptide adopts alpha-helix secondary structure from residues 309 to 334. In TFE, apoA-V(296-343) adopts an extended amphipathic alpha-helix, consistent with a role in lipoprotein binding as a component of full-length apoA-V.
authors.affiliation: Center for Prevention of Obesity, Cardiovascular Disease and Diabetes, Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, California 94609, USA.
authors.forename: Kasuen
authors.initials: K
authors.lastname: Mauldin
authors.forename: Brian L
authors.initials: BL
authors.lastname: Lee
authors.forename: Marta
authors.initials: M
authors.lastname: Oleszczuk
authors.forename: Brian D
authors.initials: BD
authors.lastname: Sykes
authors.forename: Robert O
authors.initials: RO
authors.lastname: Ryan
mesh_terms: D000595:Amino Acid Sequence; D000072040:Apolipoprotein A-V; D001054:Apolipoproteins A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008075:Lipoproteins, HDL / Q000378:metabolism / Q000502:physiology*; D008715:Methionine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011392:Proline / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017433:Protein Structure, Secondary / Q000235:genetics
chemical_list: C437704:APOA5 protein, human; D000072040:Apolipoprotein A-V; D001054:Apolipoproteins A; D008051:Lipid Bilayers; D008075:Lipoproteins, HDL; D010446:Peptide Fragments; D011392:Proline; D008715:Methionine
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