The conformation of apolipoprotein A-I in discoidal and spherical recombinant high density lipoprotein particles. 13C NMR studies of lysine ionization behavior.
To elucidate the molecular details of how high density lipoprotein (HDL) microstructure affects the conformation of apolipoprotein (apo) A-I in various classes of HDL particles, apoA-I structure in homogeneous recombinant HDL (rHDL) complexes containing palmitoyl-oleoyl phosphatidylcholine (POPC) and cholesteryl oleate has been investigated by NMR spectroscopy of [13C]lysine-labeled apoA-I. All Lys residues in rHDL apoA-I were labeled with 13C by reductive methylation, and then their ionization behavior was characterized by 13C NMR spectroscopy. Four discoidal particles were prepared to contain from 64 to 256 molecules of POPC and 2 molecules of apoA-I; their major diameters ranged from 9.3 to 12.1 nm. (13CH3)2-Lys resonances from apoA-I in discoidal complexes exhibit six distinct chemical shifts at pH 10. The various Lys have pKa values ranging from 8.3 to 10.5, indicating that they exist in different microenvironments. More than 80% of the Lys residues in small (9.3 nm) discoidal particles titrate at a significantly lower pH than in the large (12.1 nm) discoidal particles. This indicates that apoA-I has a different conformation on the differently size discs. Two spherical particles were prepared with POPC:cholesteryl oleate:apoA-I molar stoichiometries of 56:16:2 and 232:84:4 and diameters of 7.4 and 12.6 nm, respectively. On spherical rHDL, apoA-I (13CH3)2-Lys resonances exhibit five distinct chemical shifts at pH 10. The titration behavior of apoA-I Lys residues is the same in small and large spherical particles, indicating that apoA-I conformation is similar on the two particles. The Lys microenvironments indicate that the conformation of apoA-I in discoidal complexes is dependent on particle size and that these conformations are substantially different from that of apoA-I on spherical complexes. Lys microenvironments in discoidal complexes differ from that of spherical complexes by 4 to 5 ysines which titrate with relatively low pKa values on discs. This reflects apparent differences in conformation in the NH2-terminal one-third of apoA-I on discs and spheres.
Sparks
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Department of Biochemistry, Medical College of Pennsylvania, Philadelphia 19129.
Phillips
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Lund-Katz
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D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000378:metabolism; D002247:Carbon Isotopes; D003720:Densitometry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008075:Lipoproteins, HDL / Q000737:chemistry* / Q000235:genetics; D008239:Lysine; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008745:Methylation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry
D016632:Apolipoprotein A-I; D002247:Carbon Isotopes; D008075:Lipoproteins, HDL; D011994:Recombinant Proteins; D008239:Lysine