The charge and structural stability of apolipoprotein A-I in discoidal and spherical recombinant high density...

The details of how high density lipoprotein (HDL) microstructure affects the conformation and net charge of apolipoprotein (apo) A-I in various classes of HDL particles have been investigated in homogeneous recombinant HDL (rHDL) particles containing apoA-I, palmitoyl-oleoyl phosphatidylcholine (POPC) and cholesteryl oleate. Isothermal denaturation with guanidine HCl was used to monitor alpha-helix structural stability, whereas electrokinetic analyses and circular dichroism were used to determine particle charge and apoA-I secondary structure, respectively. Electrokinetic analyses show that at pH 8.6 apoA-I has a net negative charge on discoidal (POPC.apoA-I) particles (-5.2 electronic units/mol of apoA-I) which is significantly greater than that of apoA-I either free in solution or on spherical (POPC.cholesteryl oleate.apoA-I) rHDL (approximately -3.5 electronic units). Raising the POPC content (32-128 mol/ml of apoA-I) of discoidal particles 1) increases the particle major diameter from 9.3 to 12.1 nm, 2) increases the alpha-helix content from 62 to 77%, and 3) stabilizes the helical segments by increasing the free energy of unfolding (delta GD degree) from 1.4 to 3.0 kcal/mol of apoA-I. Raising the POPC content (28-58 mol/mol of apoA-I) of spherical particles 1) increases the particle diameter from 7.4 to 12.6 nm, 2) increases the percent alpha-helix from 62 to 69%, and 3) has no significant effect on delta GD degree (2.2 kcal/mol of apoA-I). This study shows that different HDL subspecies maintain particular apoA-I conformations that confer unique charge and structural characteristics on the particles. It is likely that the charge and conformation of apoA-I are critical molecular properties that modulate the metabolism of HDL particles and influence their role in cholesterol transport.