Evidence of xenon transport through the gramicidin channel: a 129Xe-NMR study.

Evidence is presented for Xe transport through the gramicidin A channel. This evidence for Xe transport through gramicidin A channels has been obtained using 129Xe-NMR spectroscopy. Three experiments were utilized. The first experiment involved monitoring the change in the chemical shift of 129Xe in the presence of increasing gramicidin A concentration, the second observed the effect on the 129Xe chemical shift with gramicidin A channels photochemically altered by UV light and the third determined the effect of gramicidin A channels blocked by Ba2+ on the 129Xe chemical shift. The results of these three experiments indicate that Xe transports through the gramicidin A channel.

Direct observation of differential UV photolytic degradation among the tryptophan residues of gramicidin A in sodium dodecyl sulfate micelles.

Gramicidin A, incorporated into sodium dodecyl sulfate micelles, was exposed to ultraviolet light and discovered by two-dimensional (TOCSY) NMR spectroscopy to undergo differential photolytic degradation. The four tryptophan residues of gramicidin A were found to be unequally sensitive to ultraviolet radiation. Tryptophan 9 was the most sensitive to ultraviolet photolysis, while tryptophan 11 was the least sensitive. Tryptophans 13 and 15 have approximately the same susceptibility to photolytic degradation by the ultraviolet light. Rate constants for the photolytic degradation of the four tryptophan residues were obtained from the dependence of the TOCSY spectrum upon the time of photolysis.

Thallium-205 NMR determination of the thermodynamics of the interaction between the thallous ion and gramicidin dimers incorporated into micelles.

A study has been made of the thermodynamics of the interaction between the thallous ion and gramicidin dimers incorporated into micelles using thallium-205 NMR spectroscopy. The chemical shift data obtained are interpreted interms of a model in which the dimer has only one tight binding site. The variation of the binding constant over the temperature range 303-323 K is used to determine the changes in enthalpy and entropy of binding giving values of -11.3 kcal/mole and -16 e.u. at 303 K, respectively.

23Na-NMR study of ion transport across vesicle membranes facilitated by phenylalanine analogs of gramicidin.

The transport of Na+ ions across phosphatidylcholine/phosphatidylglycerol large unilamellar vesicle membranes facilitated by phenylalanine analogs of gramicidin A has been studied using 23Na-NMR spectroscopy. The four analogs studied were Phe9-, Phe11-, Phe13-and Phe15-gramicidin A. These analogs were found to transport Na+ ions in the following order Phe15 > Phe13 > Phe11 > Phe9. The entropy and enthalpy of activation for the transport of Na+ ions were determined for each analog. A correlation is made between the activation enthalpies and the single channel conductance values of the analogs.

Effect of hyperbaric oxygen on sphingomyelinase D activity of brown recluse spider (Loxosceles reclusa) venom as studied by 31P nuclear magnetic resonance spectroscopy.

Hyperbaric oxygen (HBO) has been reported by some to be therapeutic for necrotic lesions induced by the venom of the brown recluse spider, Loxosceles reclusa. Others have reported no efficacy for this treatment. In this study, the effect of high pressure oxygen on an enzymatic activity of the toxin of this venom is reported. The time course for the hydrolysis of the phosphocholine ester bond of chicken egg yolk sphingomyelin, as catalyzed by brown recluse spider venom (BRSV) and venom treated with extended HBO (12 hr at 10 atmospheres), was followed by phosphorus-31 nuclear magnetic resonance spectroscopy. The venom and HBO-pretreated venom demonstrated sphingomyelinase D activity. Phospholipase C activity was not detected. The sphingomyelinase D activity of BRSV in three separate experiments was not altered by HBO. The HBO-pretreated venom, in all cases, did not exhibit an altered time course in the overall hydrolysis of the D linkage of sphingomyelin.

Effects of alanine and glycine substitution for tryptophan on the heterogeneity of gramicidin A analogs in micelles.

The effects of alanine and glycine substitution for tryptophan upon the species heterogeneity of gramicidin A analogs incorporated into SDS micelles have been investigated. The sequential replacement of the four tryptophan residues in gramicidin A at positions 15, 13, 11, and 9 with glycine showed that there was no detectable effect at position 15 but increasing heterogeneity of species in the micelles proceeding toward the interior of the micelle at position 9. The replacement of tryptophan at positions 15 and 9 with alanine was found to produce more species heterogeneity than found with glycine substitution at the same positions. An increase in the SDS concentration reduces the number of different species present in micelles. With the Gly-11, Gly-13, and Gly-15 analogs, the increase in SDS concentration results in the formation of a single species; however, for the Gly-9, Ala-9, and Ala-15 analogs, heterogeneity remains.

Sphingomyelinase D activity of brown recluse spider (Loxosceles reclusa) venom as studied by 31P-NMR: effects on the time-course of sphingomyelin hydrolysis.

The time-course for the hydrolysis of the D linkage of chicken egg yolk sphingomyelin in a Triton X-100 mixed micelle and of lysophosphotidylcholine micelles, as catalyzed by brown recluse spider venom and brown recluse spider toxin, was followed by phosphorous-31 nuclear magnetic resonance spectroscopy. The overall rate of hydrolysis of sphingomyelin in mixed micelles was found to be an order of magnitude faster than the hydrolysis of lysophosphotidylcholine. Incorporation of lysophosphotidylcholine into mixed micelles with Triton X-100 inhibited the lipase activity of brown recluse spider venom and brown recluse spider venom toxin. The effects of increased rates of overall reaction were observed with increased temperature and also with decreased ionic strength. The presence of divalent calcium ions was found to be necessary for hydrolytic activity, but only in catalytic amounts (less than 1 mM).

Soil humic-like organic compounds in prescribed fire emissions using nuclear magnetic resonance spectroscopy.

Here we present the chemical characterization of the water-soluble organic carbon fraction of atmospheric aerosol collected during a prescribed fire burn in relation to soil organic matter and biomass combustion. Using nuclear magnetic resonance spectroscopy, we observed that humic-like substances in fire emissions have been associated with soil organic matter rather than biomass. Using a chemical mass balance model, we estimated that soil organic matter may contribute up to 41% of organic hydrogen and up to 27% of water-soluble organic carbon in fire emissions. Dust particles, when mixed with fresh combustion emissions, substantially enhances the atmospheric oxidative capacity, particle formation and microphysical properties of clouds influencing the climatic responses of atmospheric aeroso. Owing to the large emissions of combustion aerosol during fires, the release of dust particles from soil surfaces that are subjected to intense heating and shear stress has, so far, been lacking.

Effects of glycine substitutions on the structure and function of gramicidin a channels.

Tryptophan residues often are found at the lipid-aqueous interface region of membrane-spanning proteins, including ion channels, where they are thought to be important determinants of protein structure and function. To better understand how Trp residues modulate the function of membrane-spanning channels, we have examined the effects of Trp replacements on the structure and function of gramicidin A channels. Analogues of gramicidin A in which the Trp residues at positions 9, 11, 13, and 15 were sequentially replaced with Gly were synthesized, and the three-dimensional structure of each analogue was determined using a combination of two-dimensional NMR techniques and distance geometry-simulated annealing structure calculations. Though Trp --> Gly substitutions destabilize the beta6.3-helical gA channel structure, it is possible to determine the structure of analogues with Trp --> Gly substitutions at positions 11, 13, and 15, but not for the analogue with the Trp --> Gly substitution at position 9. The Gly11-, Gly13-, and Gly15-gA analogues form channels that adopt a backbone fold identical to that of native gramicidin A, with only small changes in the side chain conformations of the unsubstituted residues. Single-channel current measurements show that the channel function and lifetime of the analogues are significantly affected by the Trp --> Gly replacements. The conductance variations appear to be caused by sequential removal of the Trp dipoles, which alter the ion-dipole interactions that modulate ion movement. The lifetime variations did not appear to follow a clear pattern.

Effects of phenylalanine substitutions in gramicidin A on the kinetics of channel formation in vesicles and channel structure in SDS micelles.

The common occurrence of Trp residues at the aqueous-lipid interface region of transmembrane channels is thought to be indicative of its importance for insertion and stabilization of the channel in membranes. To further investigate the effects of Trp-->Phe substitution on the structure and function of the gramicidin channel, four analogs of gramicidin A have been synthesized in which the tryptophan residues at positions 9, 11, 13, and 15 are sequentially replaced with phenylalanine. The three-dimensional structure of each viable analog has been determined using a combination of two-dimensional NMR techniques and distance geometry-simulated annealing structure calculations. These phenylalanine analogs adopt a homodimer motif, consisting of two beta6.3 helices joined by six hydrogen bonds at their NH2-termini. The replacement of the tryptophan residues does not have a significant effect on the backbone structure of the channels when compared to native gramicidin A, and only small effects are seen on side-chain conformations. Single-channel conductance measurements have shown that the conductance and lifetime of the channels are significantly affected by the replacement of the tryptophan residues (Wallace, 2000; Becker et al., 1991). The variation in conductance appears to be caused by the sequential removal of a tryptophan dipole, thereby removing the ion-dipole interaction at the channel entrance and at the ion binding site. Channel lifetime variations appear to be related to changing side chain-lipid interactions. This is supported by data relating to transport and incorporation kinetics.

Cation-binding location and hydrogen-exchange sites for gramicidin in SDS micelles using NOESY NMR.

The site of monovalent cation binding and sites of hydrogen exchange between amide protons and water molecules in the gramicidin A and Phe-1 gramicidin A channels incorporated into SDS micelles have been determined using a NOESY NMR technique. The cation-binding pocket was found to involve residues 10-15 of the peptide.

Thallium-205 and proton nuclear magnetic resonance investigation of the complexation of thallium by the ionophores monensin and nigericin.

A thallium-205 and proton nuclear magnetic resonance study of the ionophores monensin and nigericin is reported. Evidence is presented for the simultaneous existence of two forms of monensin free acid in chloroform solution, one containing a water molecule in the central cavity and one with a water molecule on the periphery, linking the ends of the monensin molecule by hydrogen bonding. There appear to be three structurally different monensin complexes of thallium, two in the salt form and one in the acid form. In free nigericin acid, both terminal hydroxyl moieties hydrogen bond to the terminal carboxylic acid oxygens; in the Na+ salt, the binding of one of the carboxylate oxygens to the metal ion allows but one of the terminal OH's to hydrogen bond strongly. Thallium-205 chemical shifts and spin-lattice relaxation times in the title complexes are discussed and compared to those in ionophores studied previously.

Species heterogeneity of Gly-11 gramicidin A incorporated into sodium dodecyl sulfate micelles.

Evidence is presented for species heterogeneity of the gly-11 analog of gramicidin A incorporated into sodium dodecyl sulfate (SDS) micelles. The evidence for species heterogeneity has been obtained using one-dimensional (1D) 1H NMR spectroscopy. The 1D spectra of the indole NH moiety of tryptophans 9, 13, and 15 show the presence of more than one species. It has been found that the heterogeneity is dependent upon the gly-11/SDS molar ratio. At high SDS concentration (i.e., gly-11/SDS of 3 mM/700 mM) the heterogeneity almost completely disappears. The temperature dependence of these 1H NMR signals suggests that the two species do not interconvert. The results of nuclear Overhauser effect spectroscopy NMR experiments indicate that one species is embedded within the micelle, while the other is nearer the aqueous interface. The importance of side chain interactions with the membrane environment in producing stable, solubilized species of small peptides in SDS micelles is illustrated.

Conformation states of gramicidin A along the pathway to the formation of channels in model membranes determined by 2D NMR and circular dichroism spectroscopy.

Gramicidin A incorporated into SDS (sodium dodecyl sulfate) micelles exists as a right-handed, N-to-N-terminal beta 6.3 helical dimer [Lomize, A. L., Orechov, V. Yu., & Arseniev, A.S. (1992) Bioorg. Khim. 18, 182-189]. In the incorporation procedure to achieve the ion channel state of gramicidin A in SDS micelles, trifluoroethanol (TFE) is used to solubilize the hydrophobic peptide before addition to the aqueous/micelle solution. The conformational transition of gramicidin A to form ion channels in SDS micelles, i.e., in TFE and 10% TFE/water, has been investigated using 2D NMR and CD spectroscopy. In neat TFE, gramicidin A was found to be monomeric and may possibly exist in an equilibrium of rapidly interconverting conformers of at least three different forms believed to be left- and/or right-handed alpha and beta 4.4 helices. It was found that the interconversion between these conformers was slowed down in 55% TFE as evident by the observation of at least three different sets of d alpha N COSY peaks although CD gave a net spectrum similar to that in neat TFE. In 10% TFE gramicidin A spontaneously forms a precipitate. The precipitated species were isolated and solubilized in dioxane where gramicidin conformers undergo very slow interconversion and could be characterized by NMR. At least seven different gramicidin A conformations were found in 10% TFE. Four of thes are the same types of double helices as previously found in ethanol (i.e., a symmetric left-handed parallel beta 5.6 double helix, an unsymmetric left-handed parallel beta 5.6 double helix, a symmetric left-handed antiparallel beta 5.6 double helix, a symmetric right-handed parallel beta 5.6 double helix); the fifth is possibly a symmetric right-handed antiparallel beta 5.6 double helix. There is also evidence for the presence of at least one form of monomeric species. Previous observation on the solvent history dependence in the ease of channel incorporation may be explained by the presence of several different folding pathways to channel formation. To test this proposal, the conformation of gramicidin A in 10% DMSO and 10% methanol was studied. In the former environment, the major form was a random coil with a minor population of double-stranded helices, while in the latter, NMR spectra indicate the presence of the same double-helical conformers as found in neat methanol.

Thallium-205 nuclear magnetic resonance study of the thallium(I)-gramicidin A association in trifluoroethanol.

Association of the thallous ion with gramicidin in 2,2,2-trifluoroethanol has been investigated by thallium-205 NMR spectroscopy. The data obtained suggest that the gramicidin dimer has two strong binding sites and one or more weak binding sites. Association constants for the strong binding sites were found to have the same value. From the temperature dependence of the strong binding site association constants, values for the association enthalpy and entropy of -2.13 +/- 0.12 kcal/mol and +5.45 +/- 0.04 eu, respectively, were obtained.

Kinetics of channel formation of gramicidins A and B in phospholipid vesicle membranes.

The thermal incorporation and channel formation of gramicidins A and B into phosphatidylcholine/phosphatidylglycerol large unilamellar vesicle membranes was studied using 23Na NMR. Delta H and delta S of activation for channel formation for gramicidin A are 11.8 kcal/mol and -11 e.u., respectively. For gramicidin B, delta H and delta S of activation are 14.6 kcal/mol and -4 e.u., respectively. Possible reasons for the differences in delta H and delta S of activation between the two analogues are discussed.

Investigation of the interaction between thallous ions and gramicidin A in dimyristoylphosphatidylcholine vesicles: a thallium-205 NMR equilibrium study.

This study reports the first direct observation of multiple occupancy of the gramicidin A channel by Tl+ ions. 205Tl NMR has been used to study the equilibrium binding of Tl+ by gramicidin A incorporated in sonicated dimyristoylphosphatidylcholine vesicles. It is shown that only multiple-channel occupancy can account for the 205Tl chemical shifts measured. The data are analyzed to yield the equilibrium association constants of 450-600 and 5-20 M-1 for the binding of the first and the second ions at 34 degrees C, respectively.

Equilibrium binding constants for the group I metal cations with gramicidin-A determined by competition studies and T1+-205 nuclear magnetic resonance spectroscopy.

The equilibrium binding constants of the Group I metal cations with gramicidin A in aqueous dispersions of lyso-PC have been determined using a combination of competitive binding with the T1+ ion and T1-205 NMR spectroscopy. The values of the binding constants at 34 degrees C are Li (32.2 M-1), Na (36.9 M-1), K (52.6 M-1), Rb (55.9 M-1), and Cs (54.0 M-1). The equilibrium binding constant for the T1+ ion at this temperature is 582 M-1. The relationships between the binding constants, the free energy of the binding process, and the cation selectivity of the gramicidin A channel are discussed.

Application of the magnetization-inversion-transfer technique to the transport of 7Li+, 23Na+, and 39K+ ions through the gramicidin channel and the M2 delta transmembrane domain of the nicotinic acetylcholine receptor.

The magnetization-inversion-transfer NMR technique has been used to determine the activation enthalpy (delta H not equal to) for the transport of the 7Li+, 23Na+, and 39K+ ions through the gramicidin channel incorporated into vesicle membranes. The activation enthalpy obtained for the overall transport of the monovalent cations was found to be in agreement with the selectivity order and single-channel ion conductance values. Furthermore, the magnetization-inversion-transfer technique was also found to be applicable to the channel formed from a synthetic peptide having the same amino acid sequence as the M2 delta transmembrane segment of the nicotinic acetylcholine receptor.

Gramicidin A/short-chain phospholipid dispersions: chain length dependence of gramicidin conformation and lipid organization.

Gramicidin-lipid interactions were investigated using diacylphosphatidylcholines that contained two identical acyl chains of varying length, between 6 and 14 carbons. The gramicidin A (gA) conformation was monitored by circular dichroism (CD) spectroscopy and high-performance size-exclusion chromatography, and the lipid organization was investigated using 31P and 1H NMR spectroscopy and negative-stain electron microscopy. Diacylphosphatidylcholine (PC) lipids with chain lengths between 4 and 8 carbons have been previously shown to have a micellar organization in aqueous solution [Lin, T.-L., et al. (1986) J. Am. Chem. Soc. 108, 3499-3507]. CD spectra of aqueous gA/lipid dispersions, at a ratio of 1:28, demonstrated that the channel conformation of gA can be readily obtained when the acyl chain length is > or = 10, but not when the chain length is < or = 7. Size-exclusion chromatography revealed that the fraction of gA that could easily be dissociated into monomers in the dispersions increased with increasing acyl chain length, in agreement with the CD results. For a chain length of 8, the results were intermediate. The formation of the channel structure was found to depend on the "solvent-history", the temperature, the gA and lipid concentrations, the gA:lipid ratio, and consequently on the method of sample preparation. 1H and 31P NMR results suggest that codispersed gA increases the size of dioctanoyl-PC aggregates, but not of dihexanoyl-PC micelles. Negative-stain electron microscopy directly supports these findings. Dihexanoyl-PC (28 mM) was able to solubilize 1 mM gA in H2O, but the gA was not in the "channel" conformation.(ABSTRACT TRUNCATED AT 250 WORDS)