The orientation of helix 4 in apolipoprotein A-I-containing reconstituted high density lipoproteins

The three-dimensional structure of the high density lipoprotein (HDL) compo nent apolipoprotein (apo) A-I and the molecular basis for its protection ag ainst coronary artery disease are unknown. In terms of discoidal HDL partic les, there has been a debate as to the orientation of the apoA-I alpha-heli ces around the disc edge. The "picket fence" model states that the alpha-he lical repeats, separated by turns, are arranged parallel to the phospholipi d acyl chains of the enclosed lipid bilayer, On the other hand, the "belt" model states that the helical segments run perpendicular to the acyl chains . To distinguish between these models, we used nitroxide spin labels presen t at various depths in the bilayer of reconstituted HDL (rHDL) to measure t he position of Trp residues in single Trp mutants of human proapoA-I. Two m utants were studied; the first contained a Trp at position 108, which was l ocated near the center of helix 4, The second contained a Trp at position 1 15, two turns along the same helix. The picket fence model predicts that th ese Trp residues should be at different depths in the bilayer, whereas the belt model predicts that they should be at similar depths. Different sized rHDL particles were produced that contained 2, 3, and >4 molecules of proap oA-I per complex. Tn each case, parallax analysis indicated that Trp-108 an d Trp-115 were present at similar depths of about 6 Angstrom from the cente r of the bilayer, consistent with helix 4 being oriented perpendicular to t he acyl chains (in agreement with the belt model). Similar experiments show ed that control transmembrane peptides were oriented parallel to the acyl c hains in vesicles, demonstrating that the method was capable of distinguish ing between the two models. This study provides one of the first experiment al measurements of the location of an apoA-I helix with respect to the bila yer edge.