Complex of Human Apolipoprotein C-1 with Phospholipid: Thermodynamic or Kinetic Stability?

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Abstract
Thermal unfolding of discoidal complexes of apolipoprotein (apo) C-1 with dimyristoyl phosphatidylcholine (DMPC) reveals a novel mechanism of lipoprotein stabilization that is based on kinetics rather than thermodynamics. Far-UV CD melting curves recorded at several heating/cooling rates from 0.047 to 1.34 K/min show hysteresis and scan rate dependence characteristic of slow nonequilibrium transitions. At slow heating rates, the apoC-1 unfolding in the complexes starts just above 25 °C and has an apparent melting temperature Tm ∼ 48 ± 1.5 °C, close to Tm = 51 ± 1.5 °C of free protein. Thus, DMPC binding may not substantially increase the low apparent thermodynamic stability of apoC-1, ΔG(25 °C) < 2 kcal/mol. The scan rate dependence of Tm and Arrhenius analysis of the kinetic data suggest an activation enthalpy Ea = 25 ± 5 kcal/mol that provides the major contribution to the free energy barrier for the protein unfolding on the disk, ΔG* ≥ 17 kcal/mol. Consequently, apoC-1/DMPC disks are kinetically but not thermodynamically stable. To explore the origins of this kinetic stability, we utilized dynode voltage measured in CD experiments that shows temperature-dependent contribution from UV light scattering of apoC-1/DMPC complexes (d ∼ 20 nm). Correlation of CD and dynode voltage melting curves recorded at 222 nm indicates close coupling between protein unfolding and an increase in the complex size and/or lamellar structure, suggesting that the enthalpic barrier arises from transient disruption of lipid packing interactions upon disk-to-vesicle fusion. We hypothesize that a kinetic mechanism may provide a general strategy for lipoprotein stabilization that facilitates complex stability and compositional variability in the absence of high packing specificity.

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