title: Molecular characterization of native and recombinant apolipoprotein A-IMilano dimer. The introduction of an interchain disulfide bridge remarkably alters the physicochemical properties of apolipoprotein A-I.
abstract: The disulfide-linked dimer of apolipoprotein A-IMilano (A-IM/A-IM), a natural Arg173-->Cys variant of apoA-I, was purified from carriers' plasma and produced in Escherichia coli. The recombinant A-IM/A-IM is identical to native A-IM/A-IM, by mass spectrometry, SDS-polyacrylamide gel electrophoresis, and isoelectric focusing. Lipid-free A-IM/A-IM undergoes concentration-dependent self-association similar to apoA-I, but at all concentrations apoA-I is more self-associated than A-IM/A-IM. Far-ultraviolet CD spectra of A-IM/A-IM reveal a highly alpha-helical structure predicted to be approximately 65% in the lipid-free and approximately 78% in the lipid-associated states, versus 43 and 73% for apoA-I. A significant loss of alpha-helix occurs below pH 3.5 and above pH 10 in both apoA-I and A-IM/A-IM; A-IM/A-IM constantly shows a higher alpha-helical content than apoA-I over the entire pH range (1.7-12.8), suggesting that hydrophobic forces stabilize the interaction between the two A-IM chains. Indeed, and differently from apoA-I, the alpha-helical content of A-IM/A-IM is minimally affected by solvent ionic strength. The aromatic side chains in both lipid-free and lipid-bound A-IM/A-IM are immobilized in a more asymmetric and hydrophobic environment than in lipid-free apoA-I, the conformation of A-IM/A-IM being instead similar to that achieved by apoA-I following interaction with lipids. The present findings prove that rA-IM/A-IM is structurally identical to the native protein; the conformation of A-IM/A-IM is remarkably different from that of apoA-I, thus possibly explaining some of the peculiar functional properties of the apoA-IMilano dimer.
authors.affiliation: Center E. Grossi Paoletti, Institute of Pharmacological Sciences, University of Milano, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Calabresi
authors.forename: G
authors.initials: G
authors.lastname: Vecchio
authors.forename: R
authors.initials: R
authors.lastname: Longhi
authors.forename: E
authors.initials: E
authors.lastname: Gianazza
authors.forename: G
authors.initials: G
authors.lastname: Palm
authors.forename: H
authors.initials: H
authors.lastname: Wadensten
authors.forename: A
authors.initials: A
authors.lastname: Hammarström
authors.forename: A
authors.initials: A
authors.lastname: Olsson
authors.forename: A
authors.initials: A
authors.lastname: Karlström
authors.forename: T
authors.initials: T
authors.lastname: Sejlitz
mesh_terms: D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I / Q000737:chemistry / Q000235:genetics*; D001120:Arginine / Q000235:genetics; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000235:genetics; D004120:Dimethyl Suberimidate; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D016632:Apolipoprotein A-I; D003432:Cross-Linking Reagents; D004220:Disulfides; D011994:Recombinant Proteins; C080631:apolipoprotein A-I Milano; D004120:Dimethyl Suberimidate; D001120:Arginine; D003545:Cysteine