Structural and functional properties of apolipoprotein A-I mutants containing disulfide-linked cysteines at positions 124 or 232.
Recombinant Cys mutants of apolipoprotein A-I (apoA-I) (A124C and A232C) have been prepared in disulfide-linked forms in order to assess the effects of unnatural covalent constraints on the folding of apoA-I in solution, its ability to bind lipids, form HDL-like particles, activate LCAT, and undergo structural adaptations to changing lipid contents. Both mutants, in dimer form, were shown to fold similarly to plasma apoA-I in solution, but had a slightly decreased alpha-helix content and no evidence of intermonomer interactions. All forms of the mutants bound to and disrupted dimyristoylphosphatidylcholine (DMPC) liposomes with similar kinetics and efficiency to plasma apoA-I, and formed reconstituted HDL (rHDL) particles with palmitoyloleoylphosphatidylcholine (POPC) in high yields at three different ratios of lipid/protein. While the monomeric mutants produced identical rHDL to plasma apoA-I, the disulfide-linked dimers had distinct particle distributions from each other and from native apoA-I. The A124C-dimer formed rHDL with diameters of 86 and 78 A, while the A232C-dimer predominantly formed 96 A rHDL. These particles, and particles containing plasma apoA-I (96 and 78 A), were purified prior to structural and functional analyses. The structural properties of particles with similar diameters were comparable, as were their reactivities with LCAT; however, their ability to undergo structural rearrangements differed. The larger rHDL particles (96 and 86 A) containing native apoA-I or A124C-dimer, rearranged into smaller 78 A particles, while the 96 A particles containing A232C-dimer were resistant to rearrangement and did not form 78 A particles. From the results, it is concluded that synthetic, random disulfide-linked dimers of apoA-I have many properties analogous to those of the naturally occurring Cys mutants, apoA-I-Milano and apoA-I-Paris, which are thought to have antiatherogenic effects in vivo. Also, the results have implications for current models of rHDL structure.
Tian
Shaomin
S
Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, 506 South Mathews Avenue, Urbana, IL 61801, USA.
Jonas
Ana
A
D016632:Apolipoprotein A-I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism; D004220:Disulfides; D004789:Enzyme Activation; D008075:Lipoproteins, HDL / Q000378:metabolism; D016296:Mutagenesis; D010316:Particle Size; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
D016632:Apolipoprotein A-I; D004220:Disulfides; D008075:Lipoproteins, HDL; D007862:Phosphatidylcholine-Sterol O-Acyltransferase; D003545:Cysteine; D004134:Dimyristoylphosphatidylcholine