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 et al., 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, The Journal of biological chemistry, 269(51), 1994, pp. 32168-32174
The disulfide-linked dimer of apolipoprotein A-I-Milano (A-I-M/A-I-M),
a natural Arg(173) --> CYS variant of apoA-I, was purified from carri
ers' plasma and produced in Escherichia coli. The recombinant A-I-M/A-
I-M is identical to native A-I-M/A-I-M, by mass spectrometry, SDS-poly
acrylamide gel electrophoresis, and isoelectric focusing. Lipid-free A
-I-M/A-I-M undergoes concentration-dependent self-association similar
to apoA-I, but at all concentrations apoA-I is more self-associated th
an A-I-M/A-I-M. Farultraviolet CD spectra of A-I-M/A-I-M reveal a high
ly alpha-helical structure predicted to be similar to 65% in the lipid
-free and similar to 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-I-M/A-I-M; A-I-M/A-I-M constantl
y shows a higher alpha-helical content than apoA-I over the entire pH
range (1.7-12.8), suggesting that hydrophobic forces stabilize the int
eraction between the two A-I-M chains. Indeed, and differently from ap
oA-I, the alpha-helical content of A-I-M/A-I-M is minimally affected b
y solvent ionic strength. The aromatic side chains in both Lipid-free
and lipid-bound A-I-M/A-I-M are immobilized in a more asymmetric and h
ydrophobic environment than in Lipid-free apoA-I, the conformation ofA
-I(M)A-I-M, being instead similar to that achieved by apoA-I following
interaction with lipids. The present findings prove that rA-I-M/A-I-M
is structurally identical to the native protein; the conformation of
A-I-M/IA-I-M is remarkably different fi om that of apoA-I, thus possib
ly explaining some of the peculiar functional properties of the apoA-I
-Milano dimer.