Mechanism of dissociation of human apolipoproteins A-I, A-11, and C from complexes with dimyristoylphosphatidylcholine as studied by thermal denaturation.

The effect of temperature on the structure of human apolipoprotein A-I (apo A-I), apo A-11, and the combined apo C fraction in the absence and presence of dimyristoylphosphatidylcholine(DMPC) has been investigated.The thermal denaturation of the apolipoproteins was monitored by circular dichroism spectroscopy. In the absence of lipid,the apolipoproteins A-I and A-I1 denature over a wide temperature range, giving van't Hoff enthalpies of 33 +/- 4 kcal/mol of apo A-I and 17.8 +/- 0.2 kcal/mol of apo A-11. These enthalpies are independent of the protein concentration, although a decrease in molar ellipticity was observed on increasing the protein concentration from 0.01 to 1 mg/mL. No effect of temperature could be observed on the combined apoC fraction because at 0.01 and 1 mg/mL the apo C's were essentially random coiled. In the presence of DMPC, thermal denaturation could be measured for apo A-I above 70-75 "C and for apo A-I1 and apo C above about 45 OC. In general,the denaturations were biphasic reactions for all apolipoproteins tested, with only a third, minor intermediate phase for apo A-I/DMPC denaturation. The two major kinetic phases are identified as an unfolding reaction of the apolipoprotein bound to the complex followed by a desorption step.The relaxation times (tau) associated with the latter step are dependent on the molecular weight of the apoprotein: when the temperature is increased from 70 to 90 OC, tau decreases from 400 to 1 min for apo A-I, while for apo A-I1 and apoC as the temperature is increased from 50 to 70 OC, tau decreases from 15 to l min. The activation energies for the desorption of apoprotein decrease with decreasing molecular weight: the values are 71 +/- 2 kcal/mol of apo A-I, 28 +/- 3 kcal/mol of apo A-11, and 22 +/- 3 kcal/mol of apo C. The thermal denaturation of apo A-I/DMPC is a thermodynamically irreversible process whereas the denaturations of apoA-II/DMPC and apo C/DMPC complexes are reversible with midpoints of 71 and 54 "C, respectively. The van't Hoff enthalpies are 16.8 +/- 0.6 kcal/mol of apo A-I1 (T < 70 "C),86 +/- 2 kcal/mol of apo A-I1 (T > 70 "C), and 22.3 +/- 0.8 kcal/mol of apo C. On the basis of the above findings, a model to describe the association and dissociation of apolipoproteins with DMPC has been derived. It is assumed that, on a molecular level, the association reaction is determined by two parameters: (1) the intrinsic rate constant describing the insertion of an apolipoprotein into a "vacancy" in the phospholipid matrix ("on rate") and (2) the probability of the colliding apoprotein molecule encountering a vacancy in the phospholipid bilayer. Alterations in either of these two parameters change the macroscopic rate constant of association.Desorption ("off rate") involves the protein leaving from a constant phospholipid environment because the perturbed adjacent lipid molecules render this process insensitive to the physical state of the remainder of the bilayer. The similarities in the van't Hoff enthalpies associated with the reversible desorption of apo A-I1 and apo C to literature values for the calorimetric enthalpies of association of these proteins with DMPC suggest that the desorption is a two-state process.