gramicidin-a and 1-2-dioleoylphosphatidylserine

We demonstrate that dynamic nuclear polarization of membrane proteins in lipid bilayers may be achieved using a novel polarizing agent: pairs of spin labels covalently bound to a protein of interest interacting at an intermolecular interaction surface. For gramicidin A, nitroxide tags attached to the N-terminal intermolecular interface region become proximal only when bimolecular channels forms in the membrane. We obtained signal enhancements of sixfold for the dimeric protein. The enhancement effect was comparable to that of a doubly tagged sample of gramicidin C, with intramolecular spin pairs. This approach could be a powerful and selective means for signal enhancement in membrane proteins, and for recognizing intermolecular interfaces.

Topics: Electron Spin Resonance Spectroscopy; Gramicidin; Lipid Bilayers; Membrane Proteins; Nuclear Magnetic Resonance, Biomolecular; Phosphatidylethanolamines; Phosphatidylserines; Protein Interaction Domains and Motifs; Spin Labels

The cyclic voltammetry behavior of a mercury-supported tethered bilayer lipid membrane (tBLM) incorporating gramicidin A was investigated in aqueous 0.1 M KCl at pH 6.8, 5.4 and 3. The distal leaflet of the lipid bilayer consisted of dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidic acid or a DOPC/cholesterol mixture. In passing from pH 6.8 to pH 3, the midpoint potential between the negative current peak, due to K(+) inflow into the spacer, and the positive current peak, due to K(+) ejection into the aqueous solution, shifts toward more positive potentials, while the separation between these two peaks decreases. This behavior is interpreted quantitatively on the basis of a model relying on tBLM structural features estimated independently in previous works. The only adjustable parameter is the rate constant for cation translocation across a potential energy barrier located in the hydrocarbon tail region. The behavior is ascribed to a dragging of the lipid headgroups adjacent to the gramicidin channel mouth toward the hydrocarbon tail region, with a resulting decrease in the negative charge of the DOPC phosphate group, or of the DOPS carboxyl group, with decreasing pH.

Topics: Electrochemistry; Gramicidin; Hydrogen-Ion Concentration; Ion Channels; Lipid Bilayers; Membrane Potentials; Mercury; Phosphatidylcholines; Phosphatidylserines; Phospholipids; Water

The potential independent limiting flux of hydrated Tl(+) ions through gramicidin (GR) channels incorporated in phospholipid monolayers self assembled on a hanging mercury-drop electrode is shown to be controlled both by diffusion and by a dehydration step. Conversely, the potential independent limiting flux of dehydrated Tl(+) ions stemming from Tl amalgam electro-oxidation is exclusively controlled by diffusion of thallium atoms within the amalgam. Modulating the charge on the polar heads of dioleoylphosphatidylserine (DOPS) by changing pH affects the limiting flux of hydrated Tl(+) ions to a notable extent, primarily by electrostatic interactions. The dipole potential of DOPS and dioleoylphosphatidylcholine (DOPC), positive toward the hydrocarbon tails, does not hinder the translocation step of Tl(+) ions to such an extent as to make it rate limiting. Consequently, incorporation in the lipid monolayer of phloretin, which decreases such a positive dipole potential, does not affect the kinetics of Tl(+) flux through GR channels. In contrast, the increase in the positive dipole potential produced by the incorporation of ketocholestanol causes the translocation step to contribute to the rate of the overall process. A model providing a quantitative interpretation of the kinetics of diffusion, dehydration-hydration, translocation, and charge transfer of the Tl(+)/Tl(0)(Hg) couple through GC channels incorporated in mercury-supported phospholipid monolayers is provided. A cut-off disk model yielding the profile of the local electrostatic potential created by an array of oriented dipoles located in the lipid monolayer along the axis of a cylindrical ion channel is developed.

Topics: Anti-Bacterial Agents; Cell Membrane; Gramicidin; Hydrogen-Ion Concentration; Ions; Ketocholesterols; Kinetics; Lipids; Mercury; Models, Chemical; Models, Statistical; Phosphatidylcholines; Phosphatidylserines; Protein Transport; Thallium; Thermodynamics; Time Factors

Hydrophobic interactions between lipid bilayers and imbedded membrane proteins couple protein conformation to the mechanical properties of the bilayer. This coupling is widely assumed to account for the regulation of membrane protein function by the membrane lipids' propensity to form nonbilayer phases, which will produce a curvature stress in the bilayer. Nevertheless, there is only limited experimental evidence for an effect of bilayer curvature stress on membrane protein structure. We show that alterations in curvature stress, due to alterations in the electrostatic energy of dioleoylphosphatidylserine bilayers, modulate the structurally well-defined gramicidin A monomer <--> dimer reaction. Maneuvers that decrease the electrostatic energy of the unperturbed bilayer promote channel dissociation; we measure the change in interaction energy. The bilayer electrostatic energy thus can affect membrane protein structure by a mechanism that does not involve the electrostatic field across the bilayer, but rather electrostatic interactions among the phospholipid head groups in each monolayer which affect the bilayer curvature stress. These results provide further evidence for the importance of mechanical interactions between a bilayer and its imbedded proteins for protein structure and function.

Topics: Gramicidin; Ion Channels; Lipid Bilayers; Osmolar Concentration; Phosphatidylserines; Protein Conformation; Static Electricity; Stress, Mechanical

The isothermal phase behavior of three gramicidin A'/phospholipid mixtures was investigated by an equilibrium Ca(2+)-binding technique. The phospholipid component was 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), or POPS/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at a constant mole ratio of 1/4. The bulk aqueous free Ca2+ concentration, [Ca2+]*f, in equilibrium with one or two gramicidin A'/phospholipid fluid phases and a small amount of the Ca (phosphatidylserine)2 gel phase, was measured as a function of composition at 20 degrees C by use of chromophoric high-affinity Ca2+ chelators. The coexistence of two gramicidin A'/phospholipid fluid phases was detected by an invariance in [Ca2+]*f over the range of compositions throughout which the two phases coexist. The compositions of the two coexisting phases are determined by the compositions at which the invariance in [Ca2+]*f begins and ends. With each of the gramicidin A'/phospholipid mixtures, we estimate that the composition of the gramicidin-poor phase is 0.03-0.04 mole fraction gramicidin A' and the composition of the gramicidin-rich phase is 0.13-0.14 mole fraction gramicidin A'. Characterization of these phases by low-angle X-ray diffraction revealed that, in each case, the gramicidin-poor phase is an L alpha phase and the gramicidin-rich phase is an HII phase. The isothermal phase behavior of gramicidin A'/POPC mixtures at approximately 23 degrees C, as determined by low-angle X-ray diffraction, was found to be similar to that of the other gramicidin A'/phospholipid mixtures.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Binding Sites; Calcium; Chelating Agents; Gramicidin; Kinetics; Magnetic Resonance Spectroscopy; Phosphatidylcholines; Phosphatidylserines; Phosphorus Radioisotopes; Reference Standards; X-Ray Diffraction

The partitioning behavior of gramicidin A' was investigated in four binary phospholipid mixtures with coexisting fluid and gel phases. The ratio of the equilibrium peptide concentration in the fluid phase to that in the gel phase (i.e., the partition coefficient, Kp) was determined by analysis of the quenching of gramicidin A' tryptophanyl fluorescence by a spin-labeled phosphatidylcholine. The partition coefficient was used as a measure of the relative solubility of gramicidin A' in the four types of gel phases analyzed. The composition of the gel phase was entirely Ca(dioleoylphosphatidylserine)2 (Ca(di18:1-PS)2), or was rich in either distearoylphosphatidylcholine (di18:0-PC), dipalmitoylphosphatidylcholine (di16:0-PC), or dimyristoylphosphatidylcholine (di14:0-PC). Except in the last case, the gel phase was depleted of gramicidin A': Kp approximately 30 when the gel phase was Ca(di18:1-PS)2 or di18:0-PC-rich, Kp approximately 10 when the gel phase was di16:0-PC-rich, and Kp approximately 1 when the gel phase was di14:0-PC-rich. The hydrophobic mismatch between the length of gramicidin A' and the length of the phospholipid acyl chains in the bulk gel phase is greatest with di18:1-PS and di18:0-PC, intermediate with di16:0-PC, and least with di14:0-PC. The Kp measurements presented here are consistent with increasing solubility of gramicidin A' in the gel phase with decreasing hydrophobic mismatch.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Dimyristoylphosphatidylcholine; Gels; Gramicidin; Liposomes; Molecular Conformation; Phosphatidylcholines; Phosphatidylserines; Phospholipids; Spectrometry, Fluorescence