ABSTRACT
The small G protein adenosine diphosphate ribosylation factor-1 (ARF1) is activated by cell membrane binding of a self-folding N-terminal domain. We present a model of the human ARF1 N-terminal peptide in planar lipid bilayers, determined from neutron lamellar diffraction and circular dichroism data with molecular modelling. This amphipathic domain lies at a shallow membrane depth, ideal for regulation of the ARF1 bio-timer by rapid, reversible membrane binding. The helical region does not elongate upon membrane binding, leaving the connecting flexible linker region's length unchanged.
Subject(s)
ADP-Ribosylation Factor 1/chemistry , Lipid Bilayers/metabolism , ADP-Ribosylation Factor 1/metabolism , Amino Acid Sequence , Circular Dichroism , Deuterium , Humans , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Models, Molecular , Neutron Diffraction , Protein Structure, TertiaryABSTRACT
Protein kinase C (PKC) and CTP:phosphocholine cytidylyltransferase (CT) are two examples of enzymes that are regulated by reversible binding to membranes, and this binding is influenced by membrane physical properties. CT activation by oxidized phosphatidylcholines was recently demonstrated and was linked to the acyl chain disordering effect of the oxidized species (Biochemistry 38, 15606). In this paper, we compare the responses of PKC and CT to an oxidized PC, and investigate the physical properties of lipid bilayers that modulate the activity of these enzymes. We show that 1-palmitoyl, 2-(11,15 dihydroxy) eicosatrienoyl PC (diOH-PAPC) caused less of an increase in the temperature of the lamellar to hexagonal II transition (T(H)) of an unsaturated PE, compared to its parent, PAPC. Using a polarity-sensitive interfacial probe, we also found evidence to suggest that this oxidized PC increases interfacial packing pressure. We found that whereas diOH-PAPC activates CT, it inhibits PKC relative to the parent PAPC. The activities of both CT and PKC are known to increase in the presence of non-lamellar forming lipids. The greater activating effect of diOH-PAPC compared with PAPC, is consistent with a stimulation of the activity of CT by negative curvature strain. However, this is not the case with PKC, for which we suggest that surface packing pressure is of prime importance.
Subject(s)
Cell Membrane/chemistry , Cell Membrane/metabolism , Choline-Phosphate Cytidylyltransferase/metabolism , Mycophenolic Acid/analogs & derivatives , Protein Kinase C/metabolism , Animals , Cell Membrane/drug effects , Dextrans , Enzyme Activation/drug effects , Fluorescent Dyes , In Vitro Techniques , Liposomes , Membrane Lipids/chemistry , Membrane Lipids/metabolism , Membranes, Artificial , Models, Biological , Oxidation-Reduction , Phospholipid Ethers/chemistry , Phospholipid Ethers/pharmacology , RatsABSTRACT
The effects of two fusion inhibitors on the lipid polymorphism of N-methylated dioleoylphosphatidylethanolamine were studied using temperature-resolved, small-angle X-ray diffraction. The inhibitory role of the tri-peptide carbobenzoxy-D-phenylalanine-L-phenylalanine-glycine and the lipid 1-lauroyl-2-hydroxy-sn-glycero-3-phosphocholine in the fusion pathway was studied, using the non-lamellar phase behaviour of the lipid as a model. We used p15EK, the N-terminal region of gp41 from feline leukaemia virus as promoter of membrane fusion, and measured the structural parameters of each observed lipid phase as a function of temperature. The fusion inhibitors were found to impede the expression of negative curvature of lipid monolayers even in the presence of fusion peptide. The results of this study are interpreted in relation to models of the membrane fusion mechanism.