Electrostatic Effects on the Stability of Discoidal High-Density Lipoproteins

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Abstract
High-density lipoproteins (HDL) remove cholesterol from peripheral tissues and thereby help to prevent atherosclerosis. Nascent HDL are discoidal complexes composed of a phospholipid bilayer surrounded by protein α-helices that are thought to form extensive stabilizing interhelical salt bridges. Earlier we showed that HDL stability, which is necessary for HDL functions, is modulated by kinetic barriers. Here we test the role of electrostatic interactions in the kinetic stability by analyzing the effects of salt, pH, and point mutations on model discoidal HDL reconstituted from human apolipoprotein C-1 (apoC-1) and dimyristoyl phosphatidylcholine (DMPC). Circular dichroism, Trp fluorescence, and light scattering data show that molar concentrations of NaCl or Na2SO4 increase the apparent melting temperature of apoC-1:DMPC complexes by up to 20 °C and decelerate protein unfolding. Arrhenius analysis shows that 1 M NaCl stabilizes the disks by δΔG* ≅ 3.5 kcal/mol at 37 °C and increases the activation energy of their denaturation and fusion by δEa ≅ δΔH* ≅ 13 kcal/mol, indicating that the salt-induced stabilization is enthalpy-driven. Denaturation studies in various solvent conditions (pH 5.7−8.2, 0−40% sucrose, 0−2 M trimethylamine N-oxide) suggest that the salt-induced disk stabilization results from ionic screening of unfavorable short-range Coulombic interactions. Thus, the dominant electrostatic interactions in apoC-1:DMPC disks are destabilizing. Comparison of the salt effects on the protein:lipid complexes of various composition reveals an inverse correlation between the lipoprotein stability and the salt-induced stabilization and suggests that short-range electrostatic interactions significantly contribute to lipoprotein stability: the better-optimized these interactions are, the more stable the complex is.