Effect of cholesterol on the charge and structure of apolipoprotein A-I in recombinant high density lipoprotein particles.
The effects of cholesterol on the conformation and net charge of apoA-I have been investigated in homogeneous recombinant high density lipoprotein (HDL) particles. ApoA-I charge and structure in discoidal recombinant HDL complexes containing palmitoyloleoylphosphatidylcholine and cholesterol have been quantitated by guanidine HCl denaturation, circular dichroism, electrokinetic analysis, and NMR spectroscopy of [13C]lysine-labeled apoA-I. In a discoidal particle containing 2 molecules of apoA-I and 160 molecules of palmitoyloleoylphosphatidylcholine, apoA-I exhibits an alpha-helix content of 75%, and the particle has a net negative surface charge of -5.2e/mol of apoA-I at pH 8.6. Addition of 2 molecules of cholesterol to this complex has no significant effect upon particle size, but slightly decreases the net charge (-5.0e) and alpha-helix content (68%) of apoA-I and enhances the stability of the helical segments, as reflected by an increase in the free energy of unfolding from 2.9 to 3.5 kcal/mol. In contrast, increasing the cholesterol content to 20 molecules/particle progressively increases particle size and apoA-I net negative charge (-6.1e), and there is a concomitant reduction in the free energy of stabilization of the alpha-helical structure in apoA-I to 2.2 kcal/mol. (13CH3)2-Lys resonances from apoA-I in discoidal recombinant HDL exhibit six chemical shifts at pH 10; these peaks originate from dimethyl-Lys residues that have pKa values ranging from 8.4 to 10.3. The titration behavior of apoA-I Lys residues is generally similar in the presence and absence of cholesterol, except that 4 Lys residues titrate at a significantly higher pH in the presence of cholesterol. These data are consistent with cholesterol having a direct effect on apoA-I conformation and charge in HDL. Structural changes of this magnitude can affect the interactions between HDL and various plasma proteins and cell surfaces. It is therefore likely that the cholesterol content of HDL plays an important role in regulating the metabolism of this lipoprotein.
Sparks
D L
DL
Department of Biochemistry, Medical College of Pennsylvania, Philadelphia 19129.
Davidson
W S
WS
Lund-Katz
S
S
Phillips
M C
MC
D016632:Apolipoprotein A-I / Q000737:chemistry*; D002784:Cholesterol / Q000737:chemistry*; D006801:Humans; D066298:In Vitro Techniques; D008075:Lipoproteins, HDL / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D010713:Phosphatidylcholines; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins
D016632:Apolipoprotein A-I; D008075:Lipoproteins, HDL; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D002784:Cholesterol; D008239:Lysine