title: Contributions of the N- and C-terminal helical segments to the lipid-free structure and lipid interaction of apolipoprotein A-I.
abstract: The tertiary structure of lipid-free apolipoprotein (apo) A-I in the monomeric state comprises two domains: a N-terminal alpha-helix bundle and a less organized C-terminal domain. This study examined how the N- and C-terminal segments of apoA-I (residues 1-43 and 223-243), which contain the most hydrophobic regions in the molecule and are located in opposite structural domains, contribute to the lipid-free conformation and lipid interaction. Measurements of circular dichroism in conjunction with tryptophan and 8-anilino-1-naphthalenesulfonic acid fluorescence data demonstrated that single (L230P) or triple (L230P/L233P/Y236P) proline insertions into the C-terminal alpha helix disrupted the organization of the C-terminal domain without affecting the stability of the N-terminal helix bundle. In contrast, proline insertion into the N terminus (Y18P) disrupted the bundle structure in the N-terminal domain, indicating that the alpha-helical segment in this region is part of the helix bundle. Calorimetric and gel-filtration measurements showed that disruption of the C-terminal alpha helix significantly reduced the enthalpy and free energy of binding of apoA-I to lipids, whereas disruption of the N-terminal alpha helix had only a small effect on lipid binding. Significantly, the presence of the Y18P mutation offset the negative effects of disruption/removal of the C-terminal helical domain on lipid binding, suggesting that the alpha helix around Y18 concealed a potential lipid-binding region in the N-terminal domain, which was exposed by the disruption of the helix-bundle structure. When these results are taken together, they indicate that the alpha-helical segment in the N terminus of apoA-I modulates the lipid-free structure and lipid interaction in concert with the C-terminal domain.
authors.affiliation: Department of Biophysical Chemistry, Kobe Pharmaceutical University, Kobe 658-8558, Japan.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Tanaka
authors.forename: Padmaja
authors.initials: P
authors.lastname: Dhanasekaran
authors.forename: David
authors.initials: D
authors.lastname: Nguyen
authors.forename: Shinya
authors.initials: S
authors.lastname: Ohta
authors.forename: Sissel
authors.initials: S
authors.lastname: Lund-Katz
authors.forename: Michael C
authors.initials: MC
authors.lastname: Phillips
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Saito
mesh_terms: D019943:Amino Acid Substitution; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D050356:Lipid Metabolism / Q000235:genetics; D008055:Lipids / Q000737:chemistry*; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000235:genetics; D013329:Structure-Activity Relationship; D013816:Thermodynamics
chemical_list: D016632:Apolipoprotein A-I; D008055:Lipids