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 α-helical structure predicted to be ∼65% in the lipid-free and ∼78% in the lipid-associated states, versus 43 and 73% for apoA-I. A significant loss of α-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 α-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 α-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.

MOLECULAR CHARACTERIZATION OF NATIVE AND RECOMBINANT APOLIPOPROTEIN A-I-MILANO DIMER - THE INTRODUCTION OF AN INTERCHAIN DISULFIDE BRIDGE REMARKABLY ALTERS THE PHYSICOCHEMICAL PROPERTIES OF APOLIPOPROTEIN-A-I / L. CALABRESI, G. VECCHIO, R. LONGHI, E. GIANAZZA, G. PALM, H. WADENSTEN, A. HAMMARSTROM, A. OLSSON, A. KARLSTROM, T. SEJLITZ, H. AGELAND, C. SIRTORI, G. FRANCESCHINI. - In: THE JOURNAL OF BIOLOGICAL CHEMISTRY. - ISSN 0021-9258. - 269:51(1994), pp. 32168-32174.

MOLECULAR CHARACTERIZATION OF NATIVE AND RECOMBINANT APOLIPOPROTEIN A-I-MILANO DIMER - THE INTRODUCTION OF AN INTERCHAIN DISULFIDE BRIDGE REMARKABLY ALTERS THE PHYSICOCHEMICAL PROPERTIES OF APOLIPOPROTEIN-A-I

L. CALABRESI
Primo
;
E. GIANAZZA;C. SIRTORI
Penultimo
;
G. FRANCESCHINI
Ultimo
1994

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 α-helical structure predicted to be ∼65% in the lipid-free and ∼78% in the lipid-associated states, versus 43 and 73% for apoA-I. A significant loss of α-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 α-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 α-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.
Settore BIO/10 - Biochimica
Settore BIO/14 - Farmacologia
1994
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/180942
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