The Carboxyl-Terminal Segment of Apolipoprotein A-V Undergoes a Lipid-Induced Conformational Change

• Published in: Biochemistry (Easton) Vol. 49; no. 23; pp. 4821 - 4826
• Main Authors: Mauldin, Kasuen, Lee, Brian L, Oleszczuk, Marta, Sykes, Brian D, Ryan, Robert O
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 15.06.2010
• Subjects: Amino Acid Sequence; Apolipoprotein A-V; Apolipoproteins A - chemistry; Apolipoproteins A - genetics; Apolipoproteins A - metabolism; Circular Dichroism; Lipid Bilayers - chemistry; Lipid Bilayers - metabolism; Lipoproteins, HDL - metabolism; Lipoproteins, HDL - physiology; Methionine - genetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Proline - genetics; Protein Binding - genetics; Protein Conformation; Protein Structure, Secondary - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi1005859

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 α-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 15N- and 15N/13C-labeled peptide in 50% TFE. Peptide backbone assignment and secondary structure prediction using TALOS+ reveal the peptide adopts α-helix secondary structure from residues 309 to 334. In TFE, apoA-V(296−343) adopts an extended amphipathic α-helix, consistent with a role in lipoprotein binding as a component of full-length apoA-V.