Membrane insertion topology of the central apolipoproteinA-I region.

PRIETO, E.D; GARDA, H.A.

Montevideo, Uruguay

Congreso; 6th Internationationa Conference of Biological Physics; 5th Southern Cone Biophysical Congres; 34Th Annual Meeting of the Argentiean Biophysical Society; 2007

ICBP 2007

Apolipoprotein A-I (apoAI) is the major protein of high-density lipoproteins (HDL) and its antiatherogenic properties are attributed to its ability to remove and delivercholesterol from peripheral tissues toward liver for catabolism and excretion.When mixed with phospholipid vesicles at the phase transition temperature, apoAI disrupts them forming discoidal HDL. At the liquid-crystalline state, apoAI binds to membranes without disrupting them, but increasing theirpermeability and promoting cholesterol desorption and exchange with other membranes or lipoprotein acceptors.ApoAI contains 243 amino acids with at least 10 homologous repeats of amphipathic a -helices. It was shown in our lab, that a central region spanning the residues 87-112 is loosely bound to lipids in discoidal HDL but itinserts into membranes when they are mixed with apoAI or HDL discs. Two amphipathic a -helices with a particular charge distribution (class "Y") are predicted in this region. We have also shown that similarly to apoAI, apeptide spanning the central region (77-120) promotes cholesterol desorption and oligomerizes in the membrane bound state without changes in the secondary structure, suggesting that the central region and itsoligomerization are essential for membrane binding.In order to obtain information on the central apoAI region conformation in lipid-free and lipid-bound states, we have used here a set of three single tryptophan mutants, each one containing the W residue at a selectedposition in the hydrophobic face of the central Y-helices (93, 104 or 108). Dependence of W-W energy transfer efficiency with protein concentration indicates that oligomerization in solution occurs through the central region.Emission properties in the lipid-bound state, and accessibility to quenchers located at different membrane depths (1-Palmitoyl-2-Stearoyl-(n-Doxyl)-sn-Glycero-3-Phosphocholine were n = 5, 10 or 14, 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phospho (Tempo) choline, or 3ß - Doxyl-5a -Cholestane) indicate an interfacial location for the position 108, and a deep insertion in the membrane for positions 93 and 104. These data support a model of insertionwith an intermolecular bundle of the central Y-helix pairs penetrating the membrane with the long helix axis perpendicular to the membrane surface.* Funded by ANPCyT, CONICET and CIC-Pcia de Bs Aires. Argentinatoward liver for catabolism and excretion.When mixed with phospholipid vesicles at the phase transition temperature, apoAI disrupts them forming discoidal HDL. At the liquid-crystalline state, apoAI binds to membranes without disrupting them, but increasing theirpermeability and promoting cholesterol desorption and exchange with other membranes or lipoprotein acceptors.ApoAI contains 243 amino acids with at least 10 homologous repeats of amphipathic a -helices. It was shown in our lab, that a central region spanning the residues 87-112 is loosely bound to lipids in discoidal HDL but itinserts into membranes when they are mixed with apoAI or HDL discs. Two amphipathic a -helices with a particular charge distribution (class "Y") are predicted in this region. We have also shown that similarly to apoAI, apeptide spanning the central region (77-120) promotes cholesterol desorption and oligomerizes in the membrane bound state without changes in the secondary structure, suggesting that the central region and itsoligomerization are essential for membrane binding.In order to obtain information on the central apoAI region conformation in lipid-free and lipid-bound states, we have used here a set of three single tryptophan mutants, each one containing the W residue at a selectedposition in the hydrophobic face of the central Y-helices (93, 104 or 108). Dependence of W-W energy transfer efficiency with protein concentration indicates that oligomerization in solution occurs through the central region.Emission properties in the lipid-bound state, and accessibility to quenchers located at different membrane depths (1-Palmitoyl-2-Stearoyl-(n-Doxyl)-sn-Glycero-3-Phosphocholine were n = 5, 10 or 14, 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phospho (Tempo) choline, or 3ß - Doxyl-5a -Cholestane) indicate an interfacial location for the position 108, and a deep insertion in the membrane for positions 93 and 104. These data support a model of insertionwith an intermolecular bundle of the central Y-helix pairs penetrating the membrane with the long helix axis perpendicular to the membrane surface.* Funded by ANPCyT, CONICET and CIC-Pcia de Bs Aires. Argentina