The effect of gramicidin A on the temperature dependence of water permeation through liposomal membranes prepared from phosphatidylcholines with different chains lengths.

The permeation of water through liposomal membranes composed of various saturated phosphatidylcholine plus gramicidin A was studied as a function of temperature. 1. The presence of gramicidin in the liposomal bilayers caused an increase in water permeability. Below the phase transition temperature this effect could be measured quite clearly in all the systems we tested, but the extent of the increase was largely dependent on the length of the hydrocarbon chains. 2. Increasing amounts of gramicidin caused a gradual disappearance of the abrupt change in the rate of water permeation near the gel-liquid crystalline phase transition temperature of dipalmitoyl phosphatidylcholine liposomes. Differential scanning calorimetry analysis of the system containing these relatively small amounts of gramicidin still showed a clear transition from the liquid crystalline to the gel state with only a slight reduction in the enthalpy change. 3. In liposomes composed of dimyristoyl, dipalmitoyl and saturated egg phosphatidylcholine there was a concomitant decrease in the activation energy of water permeation in the presence of gramicidin below and above the phase transition temperature. The activation energy for water permeation through longer chained distearoyl phosphatidylcholine liposomal bilayers was the same with or without gramicidin in the bilayer. 4. It is concluded that the ability of gramicidin to form conducting channels in a gel state bilayer depends on the thickness of the paraffin core.     

The structure and function of gramicidin A embedded in interdigitated bilayer

The effects of phase transition from normal to interdigitated lipid bilayer on the function and structure of membrane proteins were studied using linear gramicidin (gramicidin A) as a model. Interdigitated bilayer structure of dipalmitoylphosphatidylglycerol (DPPG) liposomes that was induced by atropine could not be changed notably by intercalating of gramicidin. The K+ transportation of gramicidin in both normal and interdigitated bilayer was assayed by measuring the membrane potential. Results showed that gramicidin in interdigitated bilayer exhibited lower transport capability. Intrinsic fluorescence spectrum of gramicidin in interdigitated bilayer blue-shifted 2.8 nm from the spectrum in normal bilayer, which means that interdigitation provides a more hydrophobic environment for gramicidin. Circular dichroism measurement results indicated that the conformation of gramicidin in interdigitated bilayer is not the typical beta6.3 helix as in the normal bilayer. The results suggested that the interdigitated lipid bilayer might largely affect the structure and function of membrane proteins.     

Role of thiamine thiol form in nitric oxide metabolism

In alkaline media the thiamine cyclic form is converted into a thiol form (pK(a) 9.2) with an opened thiazole ring. The thiamine thiol form releases nitric oxide from S-nitrosoglutathione (GSNO). Thiamine disulfide, mixed thiamine disulfide with glutathione, and nitric oxide are produced in the reaction. Free glutathione was recorded in small amounts. The concentration of formed nitric oxide agreed well with the concentration of degraded GSNO. The concentration of released nitric oxide was determined under anaerobic conditions spectrophotometrically by production of nitrosohemoglobin. In air, the release of nitric oxide was recorded by the production of nitrite or the oxidation of oxyhemoglobin to methemoglobin. The concentration of the thiol form in the body under physiological pH values (7.2-7.4) did not exceed 1.5-2.0%. We believe that due to the exchange reactions between the thiamine thiol form and S-nitrosocysteine protein residues, nitric oxide can be released and mixed thiamine-protein disulfides are formed. The mixed thiamine disulfides (including thiamine ester disulfides) as well as the thiamine disulfide form are quite easily reduced by low molecular weight thiols to form the thiamine cyclic form with a closed thiazole ring. A possible role of the thiamine thiol form in releasing deposited nitric oxide from low-molecular-weight S-nitrosothiols and protein S-nitrosothiols and in regulation of blood flow in the vascular bed is discussed.     

Peroxyl radicals promoted changes in water permeability through gramicidin channels in DPPC and lecithin-PC vesicles

Gramicidin incorporation to DPPC or lecithin-PC large unilamellar vesicles (LUVs) leads to pore formation that, under hyper-osmotic conditions, produces a noticeable increase in the rate of trans-membrane water flow. This pore formation is more efficient in the more fluid lecithin-PC LUVs. Exposure of these vesicles to peroxyl radicals generated in the aerobic thermolysis of 2,2'-azo-bis(2-amidinopropane) (AAPH), changes the physical properties of the bilayer (as sensed employing fluorescent probes), modifies gramicidin molecules (as sensed by the decrease in Trp fluorescence) and notably reduces the transbilayer rate of water outflow. In order to evaluate if this reduced water-transport capacity is due to changes in the membrane due to lipid-peroxidation and/or direct damage to gramicidin channels, results obtained in the oxidable vesicles (lecithin-PC) were compared to those obtained in DPPC vesicles. The data obtained show that most of the water transport efficiency loss can be ascribed to a direct disruption of gramicidin channels by AAPH derived peroxyl radicals.     

A pulse--radiolysis approach to fast reductive cleavage of a disulfide bond to uncage enzyme activity

The essential thiol of the enzyme papain has been caged by linking to an aromatic thiol. The resulting caged protein is inactive but enzymatic activity is fully restored upon chemical cleavage of the protective disulfide bond. We have exploited the chemistry of this disulfide bond to uncage papain by pulse radiolysis. We have shown that up to 10% of the enzyme activity can be restored by reductive pulse radiolysis. This approach has been tested on a small-molecule model system, and experiments on this model compound show that pulse radiolysis of the mixed cysteine-aromatic disulfide results in selective reduction of the disulfide bond to generate a thiol in 10-20% yield, consistent with the radiolytically restored activity of the caged papain quantified by the biochemical assay.     

Carboxyatractylate inhibits the uncoupling effect of free fatty acids

The ATP/ADP-antiporter inhibitors and ADP decrease the palmitate-induced stimulation of the mitochondrial respiration in the controlled state. The degree of inhibition decreases in the order: carboxyatractylate greater than bongkrekic acid, palmitoyl-CoA, ADP greater than atractylate. GDP is ineffective. The inhibiting concentration of carboxyatractylate coincides with this arresting the state 3 respiration. Carboxyatractylate inhibition decreases when the palmitate concentration increases. Stimulation of controlled respiration by FCCP or gramicidin D at any concentration of these uncouplers is carboxyatractylate-resistant, whereas that by low concentrations of DNP is partially suppressed by carboxyatractylate. These data together with observations that palmitate does not increase H+ conductance in bilayer phospholipid membranes and in cytochrome oxidase-asolectin proteoliposomes indicate that the ATP/ADP-antiporter is somehow involved in the uncoupling by low concentrations of fatty acids (or DNP), whereas that by FCCP and gramicidin D is due to their effect on the phospholipid bilayer. It is suggested that the antiporter facilitates translocation of palmitate anion across the mitochondrial membrane.     

Glutathione directly reduces an oxidoreductase in the endoplasmic reticulum of mammalian cells

The formation of disulfide bonds is an essential step in the folding of many glycoproteins and secretory proteins. Non-native disulfide bonds are often formed between incorrect cysteine residues, and thus the cell has dedicated a family of oxidoreductases that are thought to isomerize non-native bonds. For an oxidoreductase to be capable of performing isomerization or reduction reactions, it must be maintained in a reduced state. Here we show that most of the oxidoreductases are predominantly reduced in vivo. Following oxidative stress the oxidoreductases are quickly reduced, demonstrating that a robust reductive pathway is in place in mammalian cells. Using ERp57 as a model we show that the reductive pathway is cytosol-dependent and that the component responsible for the reduction of the oxidoreductases is the low molecular mass thiol glutathione. In addition, ERp57 is not reduced following oxidative stress when inhibitors of glutathione synthesis or glutathione reduction are added to cells. Glutathione directly reduces ERp57 at physiological concentrations in vitro, and biotinylated glutathione forms a mixed disulfide with ERp57 in microsomes. Our results demonstrate that glutathione plays a direct role in the isomerization of disulfide bonds by maintaining the mammalian oxidoreductases in a reduced state.     

Identification of the thiol groups in human ceruloplasmin

Human ceruloplasmin was attached to activated thiol-Sepharose via its thiol groups and was then digested with pepsin. After appropriate washings the thiol peptides were eluted by reduction and were carboxymethylated and purified by column chromatography and electrophoresis. Amino acid sequencing showed that the peptides were derived from five different areas in the molecule and together accounted for 92 residues, six of which were cysteines. Since one of the peptides contained two cysteines it seemed evident that, prior to the reductive elution of the peptides, one of these had been paired in a disulfide bridge with one of the four remaining thiol peptides present in the mixture. The disulfide was isolated and identified by digesting the immobilized protein with pepsin followed by trypsin. The second (tryptic) digestion released the disulfide peptide. Three of the true thiol peptides obtained occur in regions of sequence that have already been reported and which account for 564 of the approximately 1050 residues present in the protein. Three of them also show about 40% identity with each other, whereas no relatedness is observed with the fourth. The three related peptides are, moreover, clearly homologous to the copper-binding areas in the small blue plant and bacterial proteins plastocyanin and azurin. Homologous regions are also evident when the peptides are compared to the two sequences reported for the blue oxidase, fungal laccase, one of which contains a disulfide bridge.     

Thioredoxin-dependent Redox Regulation of Chloroplastic Phosphoglycerate Kinase from Chlamydomonas reinhardtii

In photosynthetic organisms, thioredoxin-dependent redox regulation is a well established mechanism involved in the control of a large number of cellular processes, including the Calvin-Benson cycle. Indeed, 4 of 11 enzymes of this cycle are activated in the light through dithiol/disulfide interchanges controlled by chloroplastic thioredoxin. Recently, several proteomics-based approaches suggested that not only four but all enzymes of the Calvin-Benson cycle may withstand redox regulation. Here, we characterized the redox features of the Calvin-Benson enzyme phosphoglycerate kinase (PGK1) from the eukaryotic green alga Chlamydomonas reinhardtii, and we show that C. reinhardtii PGK1 (CrPGK1) activity is inhibited by the formation of a single regulatory disulfide bond with a low midpoint redox potential (−335 mV at pH 7.9). CrPGK1 oxidation was found to affect the turnover number without altering the affinity for substrates, whereas the enzyme activation appeared to be specifically controlled by f-type thioredoxin. Using a combination of site-directed mutagenesis, thiol titration, mass spectrometry analyses, and three-dimensional modeling, the regulatory disulfide bond was shown to involve the not strictly conserved Cys²²⁷ and Cys³⁶¹. Based on molecular mechanics calculation, the formation of the disulfide is proposed to impose structural constraints in the C-terminal domain of the enzyme that may lower its catalytic efficiency. It is therefore concluded that CrPGK1 might constitute an additional light-modulated Calvin-Benson cycle enzyme with a low activity in the dark and a TRX-dependent activation in the light. These results are also discussed from an evolutionary point of view.     

Astrocytes provide cysteine to neurons by releasing glutathione

Cysteine is the rate-limiting precursor of glutathione synthesis. Evidence suggests that astrocytes can provide cysteine and/or glutathione to neurons. However, it is still unclear how cysteine is released and what the mechanisms of cysteine maintenance by astrocytes entail. In this report, we analyzed cysteine, glutathione, and related compounds in astrocyte conditioned medium using HPLC methods. In addition to cysteine and glutathione, cysteine-glutathione disulfide was found in the conditioned medium. In cystine-free conditioned medium, however, only glutathione was detected. These results suggest that glutathione is released by astrocytes directly and that cysteine is generated from the extracellular thiol/disulfide exchange reaction of cystine and glutathione: glutathione + cystine<-->cysteine + cysteine-glutathione disulfide. Conditioned medium from neuron-enriched cultures was also assayed in the same way as astrocyte conditioned medium, and no cysteine or glutathione was detected. This shows that neurons cannot themselves provide thiols but instead rely on astrocytes. We analyzed cysteine and related compounds in rat CSF and in plasma of the carotid artery and internal jugular vein. Our results indicate that cystine is transported from blood to the CNS and that the thiol/disulfide exchange reaction occurs in the brain in vivo. Cysteine and glutathione are unstable and oxidized to their disulfide forms under aerobic conditions. Therefore, constant release of glutathione by astrocytes is essential to maintain stable levels of thiols in the CNS.     

Interaction of ions and water in gramicidin A channels: streaming potentials across lipid bilayer membranes

For very narrow channels in which ions and water cannot overtake one another (single-file transport), electrokinetic measurements provide information about the number of water molecules within a channel. Gramicidin A is believed to form such narrow channels in lipid bilayer membranes. In 0.01 and 0.1 M solutions of CsCl, KCL, and NaCl, streaming potentials of 3.0 mV per osmolal osmotic pressure difference (created by urea, glycerol, or glucose) appear across gramicidin A-treated membranes. This implies that there are six to seven water molecules within a gramicidin channel. Electroosmotic experiments, in which the water flux assoicated with current flow across gramicidin-treated membranes is measured, corroborate this result. In 1 M salt solutions, streaming potentials are 2.35 mV per osmolal osmotic pressure difference instead of 3.0 mV. The smaller value may indicate multiple ion occupancy of the gramicidin channel at high salt concentrations. Apparent deviations from ideal cationic selectivity observed while attempting to measure single-salt dilution potentials across gramicidin-treated membranes result from streaming potential effects.     

Antigenicities of stem bromelain. Contribution of three-dimensional structure and individual amino acid residues

The contribution of three-dimensional structure and individual amino acid residues to the antigenicities of macromolecular protein was investigated for a thiol protease stem bromelain as antigen. The extent of the participation was demonstrated by a decrease in antigenicity when the enzyme was denatured in 8 M urea before and after reductive cleavage of intrapeptide disulfide bonds or modified in particular amino acid residues. The results showed that the enzyme treated with 8 M urea without reductive cleavage of disulfide bonds preserved about 90% of antigenicity to antibodies against native stem bromelain, while the enzyme denatured after the reductive cleavage of disulfide bonds brought about almost 80% disappearance of the antigenicity. Modification of individual amino acid side chains revealed that lysine was the most immunodominant amino acid, showing 2.5% contribution per residue, and tyrosine followed with 1.2%. However, acidic amino acids such as flutamic and aspartic acids were found to be as low as 0.3%, and tryptophan was 0.2%. These data suggest that most of the antigenic determinants of stem bromelain are of the steric conformation in which lysine and/or tyrosine are most frequently involved as immunodominant amino acids.     

Gramicidin structure and disposition in highly curved membranes

With a view to deciphering aspects of the mechanism of membrane protein crystallization in lipidic mesophases (in meso crystallization), an examination of the structure and disposition of the pore-forming peptide, gramicidin, in the lipidic cubic phase was undertaken. At its simplest, the cubic phase consists of lipid and water in the form of a molecular 'sponge.' The lipid exists as a continuous, highly curved bilayer that divides the aqueous component into two interpenetrating but non-contacting channels. In this study, we show that gramicidin reconstitutes into the lipid bilayer of the cubic phase and that it adopts the channel, or helical dimer, conformation therein. Fluorescence quenching with brominated lipid was used to establish the bilayer location of the peptide. Electronic absorption and emission spectroscopies corroborated this finding. Peptide conformation in the cubic phase membrane was determined by circular dichroism. The identity and microstructure of the mesophases, and their capacity to accommodate gramicidin and other system components (sodium dodecyl sulfate, trifluoroethanol), was established by small-angle X-ray diffraction. Beyond a limiting concentration, gramicidin destabilized the cubic phase in favor of the inverted hexagonal phase. While gramicidin remained bilayer bound as membrane thickness changed, its conformation responded to the degree of bilayer mismatch with the hydrophobic surface of the peptide. These findings support the hypothesis that reconstitution into the lipid bilayer is an integral part of the in meso crystallization process as applied to membrane proteins. They also suggest ways for improving the process of membrane protein crystallogenesis.     

The action of gramicidin S on the ionic permeability of bilayer lipid membranes

The effect of cyclic decapeptide of gramicidin S on electrical conductivity of bilayer lipid membranes has been studied. The integral conductivity of bilayer has been shown to increase with the growth of antibiotic concentration. The integral conductivity increase occurs as series of conductivity discrete leaps, differing in amplitude from fluctuations of conductivity caused by linear gramicidins. In the series of selectivity of bilayer membranes for cations of alkaline metals the rubidium ion is before the cesium ion. This is the only difference between this series and the series of relative ionic mobility series of cations of alkaline metals in water solutions.     

Protein stability and conformational rearrangements in lipid bilayers: linear gramicidin, a model system.

The replacement of four tryptophans in gramicidin A by four phenylalanines (gramicidin M) causes no change in the molecular fold of this dimeric peptide in a low dielectric isotropic organic solvent, but the molecular folds are dramatically different in a lipid bilayer environment. The indoles of gramicidin A interact with the anisotropic bilayer environment to induce a change in the molecular fold. The double-helical fold of gramicidin M, as opposed to the single-stranded structure of gramicidin A, is not compatible with ion conductance. Gramicidin A/gramicidin M hybrid structures have also been prepared, and like gramicidin M homodimers, these dimeric hybrids appear to have a double-helical fold, suggesting that a couple of indoles are being buried in the bilayer interstices. To achieve this equilibrium structure (i.e., minimum energy conformation), incubation at 68 degrees C for 2 days is required. Kinetically trapped metastable structures may be more common in lipid bilayers than in an aqueous isotropic environment. Structural characterizations in the bilayers were achieved with solid-state NMR-derived orientational constraints from uniformly aligned lipid bilayer samples, and characterizations in organic solvents were accomplished by solution NMR.     

2H-nuclear magnetic resonance investigations on phospholipid acyl chain order and dynamics in the gramicidin-induced hexagonal HII phase.

The following results are reported in this paper: The interaction of gramicidin with [11,11-2H2]dioleoylphosphatidylcholine (DOPC) and [11,11-2H2]dioleoylphosphatidylethanolamine (DOPE) at different stages of hydration was studied by 2H- and 31P-nuclear magnetic resonance. In the L alpha phase in excess water the acyl chains of phosphatidylethanolamine (PE) are more ordered than phosphatidylcholine (PC) most likely as the result of the lower headgroup hydration of the former lipid. In excess water gramicidin incorporation above 5 mol % in DOPC causes a bilayer----hexagonal HII phase change. In the HII phase acyl chain order is virtually unaffected by gramicidin but the peptide restricts the fast chain motions. At low water content gramicidin cannot induce the HII phase but it markedly decreases chain order in the DOPC bilayer. Increasing water content results in separation between a gramicidin-poor and a gramicidin-rich L alpha phase with decreased order of the entire lipid molecule. Further increase in hydration reverts at low gramicidin contents the phase separation and at high gramicidin contents results in a direct change of the disordered lamellar to the hexagonal HII phase. Gramicidin also promotes HII phase formation in the PE system but interacts much less strongly with PE than with PC. The results support our hypothesis that gramicidin, by a combination of strong intermolecular attraction forces and its pronounced cone shape, both involving the four tryptophans at the COOH-terminus, has a strong tendency to organize, with the appropriate lipid, in intramembranous cylindrical structures such as is found in the HII phase.     

Theoretical analysis of hydrophobic matching and membrane-mediated interactions in lipid bilayers containing gramicidin.

We present a quantitative analysis of the effects of hydrophobic matching and membrane-mediated protein-protein interactions exhibited by gramicidin embedded in dimyristoylphosphatidylcholine (DMPC) and dilauroylphosphatidylcholine (DLPC) bilayers (Harroun et al., 1999. Biophys. J. 76:937-945). Incorporating gramicidin, at 1:10 peptide/lipid molar ratio, decreases the phosphate-to-phosphate (PtP) peak separation in the DMPC bilayer from 35.3 A without gramicidin to 32.7 A. In contrast, the same molar ratio of gramicidin in DLPC increases the PtP from 30.8 A to 32.1 A. Concurrently, x-ray in-plane scattering showed that the most probable nearest-neighbor separation between gramicidin channels was 26.8 A in DLPC, but reduced to 23.3 A in DMPC. In this paper we review the idea of hydrophobic matching in which the lipid bilayer deforms to match the hydrophobic surface of the embedded proteins. We use a simple elasticity theory, including thickness compression, tension, and splay terms to describe the membrane deformation. The energy of membrane deformation is compared with the energy cost of hydrophobic mismatch. We discuss the boundary conditions between a gramicidin channel and the lipid bilayer. We used a numerical method to solve the problem of membrane deformation profile in the presence of a high density of gramicidin channels and ran computer simulations of 81 gramicidin channels to find the equilibrium distributions of the channels in the plane of the bilayer. The simulations contain four parameters: bilayer thickness compressibility 1/B, bilayer bending rigidity Kc, the channel-bilayer mismatch Do, and the slope of the interface at the lipid-protein boundary s. B, Kc, and Do were experimentally measured; the only free parameter is s. The value of s is determined by the requirement that the theory produces the experimental values of bilayer thinning by gramicidin and the shift in the peak position of the in-plane scattering due to membrane-mediated channel-channel interactions. We show that both hydrophobic matching and membrane-mediated interactions can be understood by the simple elasticity theory.     

Membrane dipole potential modulates proton conductance through gramicidin channel: movement of negative ionic defects inside the channel.

The effect of membrane dipole potential on gramicidin channel activity in bilayer lipid membranes (BLMs) was studied. Remarkably, it appeared that proton conductance of gramicidin A (gA) channels responded to modulation of the dipole potential oppositely as compared with gA alkali metal cation conductance. In particular, the addition of phloretin, known to reduce the membrane dipole potential, resulted in a decrease in gA proton conductance, on one hand, and an increase in gA alkali metal conductance, on the other hand, whereas 6-ketocholestanol, the agent raising the membrane dipole potential, provoked an increase in gA proton conductance as opposed to a decrease in the alkali metal cation conductance. The peculiarity of the 6-ketocholestanol effect consisted in its dependence on the H(+) concentration. The experiments with the impermeant dipolar compound, phloridzin, showed that the response of proton transport through gramicidin channels to varying the membrane dipole potential did not change qualitatively if the dipole potential of only one monolayer or both monolayers of the BLM was altered. In contrast to gA proton conductance, the single-channel lifetime changed similarly with varying the membrane dipole potential, regardless of the kind of permeant cations (protons or potassium ions). The results of this study could be tentatively accounted for by an assumption that one of the rate-limiting steps of proton conduction through gramicidin channels represents, in fact, movement of negatively charged species (negative ionic defects) across a membrane.     

The oxidative folding of proteins by disulfide plus thiol does not correlate with redox potential

The rate and yield of oxidative renaturation of reduced RNase A has been studied as a function of [-S-S-]/[-SH]. The principal conclusion of these studies is that rates and yields of oxidative renaturation are strongly dependent on the low mol. wt disulfide/thiol ratio. The relationships are complex and do not parallel the redox potential of the system. The present results are consistent with earlier findings on other proteins, and lead us to believe that the above conclusion is general. Kinetic studies of oxidative renaturation should recognize and account for the dependence of reaction rate and extent on the disulfide/thiol ratio. This ratio can change substantially over the course of a reaction, either due to stoichiometric transfer of disulfide to protein, and/or adventitious air oxidation of thiols. Failure to account for changes in the disulfide/thiol ratio may compromise the interpretation of such experiments.