Showing papers by "Richard M. Epand published in 1993"

Effect of end group blockage on the properties of a class A amphipathic helical peptide

Abstract: In a recent classification of biologically active amphipathic alpha-helixes, the lipid-associating domains in exchangeable plasma apolipoproteins have been classified as class A amphipathic helixes (Segrest, J.P., De Loof, H., Dohlman, J.G., Brouillette, C.G., Anantharamaiah, G.M. Proteins 8:103-117, 1990). A model peptide analog with the sequence, Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu Ala Phe (18A), possesses the characteristics of a class A amphipathic helix. The addition of an acetyl group at the alpha-amino terminus and an amide at the alpha-carboxyl terminus, to obtain Ac-18A-NH2, produces large increases in helicity for the peptide both in solution and when associated with lipid (for 18A vs Ac-18A-NH2, from 6 to 38% helix in buffer and from 49 to 92% helix when bound to dimyristoyl phosphatidylcholine in discoidal complexes). Blocking of the end-groups of 18A stabilizes the alpha-helix in the presence of lipid by approximately 1.3 kcal/mol. There is also an increase in the self-association of the blocked peptide in aqueous solution. The free energy of binding to the PC-water interface is increased only by about 3% (from -8.0 kcal/mol for 18A to -8.3 kcal/mol for Ac-18A-NH2). The Ac-18A-NH2 has a much greater potency in raising the bilayer to hexagonal phase transition temperature of dipalmitoleoyl phosphatidylethanolamine than does 18A. In this regard Ac-18A-NH2 more closely resembles the behavior of the apolipoprotein A-I, which is the major protein component of high-density lipoprotein and a potent inhibitor of lipid hexagonal phase formation. The activation of the plasma enzyme lecithin: cholesterol acyltransferase by the Ac-18A-NH2 peptide is greater than the 18A analog and comparable to that observed with the apo A-I. In the case of Ac-18A-NH2, the higher activating potency may be due, at least in part, to the ability of the peptide to micellize egg PC vesicles.

Mechanism of activation of protein kinase C: roles of diolein and phosphatidylserine.

Abstract: We studied the roles of lipid concentration, phosphatidylserine (PS), and diolein (DG) contents, as well as Ca2+ concentration, on the partitioning of protein kinase C (PKC) between aqueous and membrane environments as well as the relationship of this partitioning to the activation of the enzyme. Physiological concentrations of 1 mol % DG increased the apparent binding constant of PKC to the 3:1 PC/PS membrane 500 times. This increase was proportional to the mol % DG. Over 50% PKC was bound to that membrane at micromolar concentrations of Ca2+ and physiologically relevant total concentration of lipid only when 1 mol % DG was included. PKC bound either to PS alone or to PS and DG was enzymatically competent; however, the rate of phosphorylation was doubled in the presence of 1 mol % diolein. The dependence of PKC binding on the mol % PS was highly sigmoidal. The Hill coefficient was in the range of 4-6, with the higher values found at the lower lipid concentrations. These results suggest that the observed apparent cooperativity is due, at least in part, to the change in dimensionality when PKC binds to the membrane.

Direct evidence for the partial dehydration of phosphatidylethanolamine bilayers on approaching the hexagonal phase

Abstract: X-ray diffraction studies on oriented multilayers of 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) in the lamellar gel (L beta) and inverted hexagonal (HII) phases at various temperatures (5-50 degrees C) and relative humidities (0-100%) are reported. One-dimensional electron density profiles of the L beta phase bilayers were constructed to a resolution of better than 4 A using direct methods to solve for the phase problem. In addition, the electron density profiles were fitted favorably using a model in which the atomic groups were assumed to be Gaussian distributed [Wiener, M. C., & White, S. H. (1992) Biophys. J. 61, 434-447]. The X-ray data clearly demonstrate that, at 100% relative humidity (RH), POPE samples exist in two distinct L beta phases, differing primarily in the amount of water between the lamellae. As the hexagonal phase transition temperature is approached, 100% RH POPE samples partially dehydrate, releasing approximately 5 water molecules per phospholipid and experiencing on average a 3-A decrease in repeat spacing. The lower temperature hydrated L beta phase POPE electron density distribution resembles that obtained from the L beta phase 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayers and is unlike the partially dehydrated POPE bilayers.

Role of prenylation in the interaction of the a-factor mating pheromone with phospholipid bilayers.

Abstract: We have studied the interaction between phospholipids and a-factor (YIIKGVFWDPAC-[Farn]OMe), S-alkylated forms of a-factor with the farnesyl group substituted by methyl, hexadecanyl, or benzyl groups, and truncated forms of this lipopeptide. Circular dichroism studies suggest that, despite its lack of farnesylation, S-methyl-a-factor is incorporated into vesicles of dimyristoylphosphatidylcholine in a conformation similar to that which a-factor adopts in this membrane. However, studies of the intrinsic fluorescence of the Trp residues of these peptides indicate that this residue is more deeply imbedded into the bilayer in the case of the farnesylated peptide. The a-factor is more effective in raising the bilayer to the hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine than is the S-methyl-a-factor. This bilayer-stabilizing ability is also reflected in a-factor inhibiting leakage from vesicles of N-methyldioleoylphosphatidylethanolamine. Studies on a-factor analogs permit the conclusion that the bilayer-stabilizing effect of a-factor is not solely a consequence of its greater partitioning into the membrane but is also a consequence of the degree of penetration into the bilayer and the specific conformation of the peptide at the membrane interface. These results indicate that the farnesyl group alone, in the absence of cellular factors, bestows a particular physical interaction with membranes.