Equilibrium binding constants for Tl+ with gramicidins A, B and C in a lysophosphatidylcholine environment determined by 205Tl nuclear magnetic resonance spectroscopy.

Nuclear Magnetic Resonance (NMR) 205Tl spectroscopy has been used to monitor the binding of Tl+ to gramicidins A, B, and C packaged in aqueous dispersions of lysophosphatidylcholine. For 5 mM gramicidin dimer in the presence of 100 mM lysophosphatidylcholine, only approximately 50% or less of the gramicidin appears to be accessible to Tl+. Analysis of the 205Tl chemical shift as a function of Tl+ concentration over the 0.65-50 mM range indicates that only one Tl+ ion can be bound by gramicidin A, B, or C under these experimental conditions. In this system, the Tl+ equilibrium binding constant is 582 +/- 20 M-1 for gramicidin 1949 +/- 100 M-1 for gramicidin B, and 390 +/- 20 M-1 for gramicidin C. Gramicidin B not only binds Tl+ more strongly but it is also in a different conformational state than that of A and C, as shown by Circular Dichroism spectroscopy. The 205Tl NMR technique can now be extended to determinations of binding constants of other cations to gramicidin by competition studies using a 205Tl probe.     

The occupancy of ions in the K+ selectivity filter: charge balance and coupling of ion binding to a protein conformational change underlie high conduction rates

Potassium ions diffuse across the cell membrane in a single file through the narrow selectivity filter of potassium channels. The crystal structure of the KcsA K+ channel revealed the chemical structure of the selectivity filter, which contains four binding sites for K+. In this study, we used Tl+ in place of K+ to address the question of how many ions bind within the filter at a given time, i.e. what is the absolute ion occupancy? By refining the Tl+ structure against data to 1.9A resolution with an anomalous signal, we determined the absolute occupancy of Tl+. Then, by comparing the electron density of Tl+ with that of K+, Rb+ and Cs+, we estimated the absolute occupancy of these three ions. We further analyzed how the ion occupancy affects the conformation of the selectivity filter by analyzing the structure of KcsA at different concentrations of Tl+. Our results indicate that the average occupancy for each site in the selectivity filter is about 0.63 for Tl+ and 0.53 for K+. For K+, Rb+ and Cs+, the total number of ions contained within four sites in the selectivity filter is about two. At low concentrations of permeant ion, the number of ions drops to one in association with a conformational change in the selectivity filter. We conclude that electrostatic balance and coupling of ion binding to a protein conformational change underlie high conduction rates in the setting of high selectivity.     

Effect of thallium ions on gramicidin-induced conductance of muscle fiber membranes

Conductance of single fibres from m. ileofibularis of Rana esculenta was studied in isotonic K2SO4 solution under constant current conditions using the double sucrose gap method. It was found that Tl+ (at concentrations 5, 10, and 20 mM) blocked K+ currents in the gramicidin channel. The decrease of K+ conductance caused by Tl+ was associated with the changes of the membrane potential. Both the decrease of K+ conductance and value of permeability ratio (PTl/PK) found from the membrane potential changes depended on Tl+ concentration in the bathing solution. No effect of Tl+ on the potassium channels was registered in the absence of gramicidin channels. The Tl+ block described here proves the existence of Tl+ ion binding within gramicidin channels of the muscle membrane and interactions among ions in the channels.     

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.     

Difference in association of Tl(I) with gramicidin A and gramicidin B in trifluoroethanol determined by Tl-205 NMR

The thallium-205 chemical shift was determined as a function of temperature for the thallium(I) complexes of gramicidin A and gramicidin B in 2,2,2-trifluoroethanol. From the difference in magnitude of the induced chemical shift it was determined that gramicidin B does not bind the Tl(I) ion as well as does gramicidin A. This result may explain the lower single-channel conductance of gramicidin B relative to gramicidin A. Cabon-13 NMR studies strongly indicate that the binding site for gramicidin A and B is at teh tryptophan end of the molecule and that replacement of tryptophan residue at position 11 in gramicidin A with a phenylalanine to form gramicidin B produces a significant structural change at the tryptophan end of the molecule, but has little effect on the N-terminus.     

Estimation of Na+ dwell time in the gramicidin A channel. Na+ ions as blockers of H+ currents

The permeation of Na+ through gramicidin A channels shows a simple saturation with increasing Na+ concentration that can be described by two different models. The first model assumes that one Na+ binds to the channel with high affinity (approximately 30 M-1) and that conduction occurs by a 'knock-on' mechanism requiring double occupancy of the channel; the other model assumes that Na+ binding is of low affinity (less than 1 M-1), and that double occupancy of the channel is rare. NMR measurements have shown tight Na+ binding, favoring the first model, but measurements of flux ratios and water transport support the second model. We present here a relatively model-independent measurement of the dwell time of Na+ inside the channel, in which we characterize the fluctuations in H+ current through the channel induced by 'block' from the more slowly permeating Na+ ions. The mean Na+ dwell time inside the channel is estimated to be approximately 10 ns at a membrane potential of 200 mV. This result is inconsistent with tight Na+ binding, thus favoring the second model.     

The effects of calcium ions on double helical forms of gramicidin

The effects of binding calcium ions to the double helical forms of gramicidin present in methanol solution were examined using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. It was found that calcium ions principally alter the relative composition of the equilibrium mixture of gramicidin conformers present in the solvent. In the absence of calcium, both parallel and antiparallel double helices are present. However, the addition of small amounts of Ca²⁺ shifts the equilibrium towards the left-handed parallel double helical form. This conformational change prevents monovalent cations (caesiums) from binding to the gramicidin double helix, and even converts the shorter, wider anti-parallel double helical form normally produced in the presence of caesium into the longer, narrower parallel double helical form. Furthermore, a temperature study showed that calcium ions tend to stabilize this form relative to the ion-free forms. The conformation of gramicidin is further changed, becoming a disordered structure, when the concentration of Ca²⁺ is raised. Thus, the binding of divalent calcium ions has a number of dramatic effects on the conformations of gramicidin present in solution.     

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.     

Thermodynamic parameters for the binding of divalent cations to gramicidin A incorporated into a lipid environment by Tl-205 nuclear magnetic resonance.

Thermodynamic parameters, enthalpy and entropy, for the binding of the divalent cations, Mg+2, Ca+2, Sr+2, Ba+2, and Cd+2, to gramicidin A, incorporated into lysophosphatidylcholine, have been determined using a combination of Tl-205 nuclear magnetic resonance spectroscopy and competition binding. The binding process is thermodynamically driven by the enthalpy and not the entropy. The enthalpy values are related to the process involving the transfer of cations from an aqueous environment to an amide environment. A comparison is made between the thermodynamic parameters for the binding of monovalent and divalent cations to gramicidin A to illustrate the channel blocking ability of the divalent cations with respect to monovalent cation transport.     

Characterization of the monovalent cation activator binding site of S-adenosylmethionine synthetase by 205Tl NMR of enzyme-bound Tl+

The structure of the binding site for the monovalent cation activator of S-adenosylmethionine (AdoMet) synthetase from Escherichia coli has been characterized by 205Tl NMR of enzyme-bound Tl+. The chemical shift of the enzyme-Tl+ complex is 176 ppm downfield from aquo Tl+, a shift which is typical only of Tl+ complexes with solely oxygen ligands. The 205Tl resonance shifts upfield to 85 ppm in the enzyme-Mg(II)-Tl+ complex, to 38 ppm in the enzyme-Tl+-AdoMet complex and to 34 ppm in the enzyme-Tl+-AdoMet-Mg(II) complex. The 205Tl chemical shift of enzyme-bound Tl+ was not altered by binding of either methionine, or the Mg(II)-ATP analog Mg(II)-adenyl-5'-yl imidodiphosphate, or Mg(II)-pyrophosphate to the enzyme-Tl+-Mg(II) complex. The NMR data suggest that the substrates or products of the enzyme do not coordinate to the monovalent cation activator and imply that monovalent cation activation results from alterations in protein conformation.     

23Na-nuclear magnetic resonance investigation of gramicidin-induced ion transport through membranes under equilibrium conditions.

A technique for investigating the gramicidin-facilitated transport of Na+ ions across lipid bilayers of large unilamellar vesicles under the condition of ionic equilibrium has been developed using a combination of heat incubation of the gramicidin with the vesicles and 23Na-nuclear magnetic resonance (NMR) spectroscopy. Isolation of the two 23Na-NMR signals from the intra- and extravesicular Na+ with the shift reagent, dysprosium (III) tripolyphosphate, allows the equilibrium flux of Na+ through the gramicidin channels to be detected and treated as a two-site exchange process. This study indicates that the transport of Na+ through gramicidin channels is second order with respect to the gramicidin concentration.     

Nuclear magnetic resonance of 23Na ions interacting with the gramicidin channel.

Basic nuclear magnetic resonance (NMR) features of 23Na ions bound to the gramicidin channel (packaged into lecithin liposomes) were studied. The first binding constant K1 of Na+ was not significantly dependent on channel models employed. With the two-identical-site model (Model I), K1 was 13.7 (+/- 1.4) molal-1 (in the activity basis) at 25 degrees C; when the binding of a third ion was included (Model II), it was 13.0 (+/- 2.0) molal-1. The second binding constant K2 was model dependent; it was 1.6 (+/- 0.2) and 3-4 molal-1 for Models I and II, respectively. The rate constants, k-1 and k-2, of Na+ for exit from singly and doubly loaded channels, respectively, were 8 X 10(5) s-1 less than or equal to k-1 less than or equal to 3 X 10(6) s-1 and 8 X 10(5) s-1 less than or equal to k-2 less than or equal to 1.0 X 10(7) s-1 at 25 degrees C; the lower bound represents a rough approximation of k-1. The ratio k-2/k-1 was greater than one and did not greatly exceed 20. From the competition experiment, K1 of T1+ was 5.7 (+/- 0.6) X 10(2) molal-1. The longitudinal relaxation time T1 of bound 23Na in the state of single occupancy (T 1B sing) was virtually independent of models, 0.56 (+/- 0.03) and 0.55 (+/- 0.04) ms at 25 degrees C for Models I and II, respectively. For the state of double occupancy, T1 of bound 23Na (T 1B doub) was model dependent: 0.27 (+/- 0.01) and 0.4-0.6 ms for Models I and II. The correlation time tau c of bound 23Na was 2.2 (+/- 0.2) ns at 25 degrees C for single occupancy; tau c for double occupancy was not significantly different from this value. The estimated tau c was found to involve no appreciable contribution of the exchange of 23Na between the channel and the bulk solution. Thé quadrupole coupling constant chi was 1.0 (+/- 0.1) MHz for 23Na in single occupancy; chi for double occupancy was 0.9-1.4 MHz, depending on models. A lower bound of the average quadrupole coupling constant chi alpha was 0.13-0.26 MHz at 25 degrees C for 23Na in single occupancy; this value represents a rough approximation of chi alpha at this temperature. An argument based on the estimated chi alpha and the known conformation of the gramicidin channel suggests that the binding site is a small domain near the channel end.(ABSTRACT TRUNCATED AT 400 WORDS)     

The divalent cation-binding sites of gramicidin A transmembrane ion-channel.

The conductance of the gramicidin A single channels in glycerolmonooleate membranes is strongly reduced in the presence of Mn2+ cations. The nmr experiments were performed for N-terminal to N-terminal gramicidin A dimer formed by two right-handed single-stranded helixes incorporated into the sodium dodecyl sulfate micelles in the presence of Mn2+ ions. Dependence of the nonselective spin-lattice relaxation rates of the gramicidin A protons on Mn2+ concentration was analyzed to determine coordinates of the divalent cation binding sites. It is inferred that Mn2+ ions are bound at the channel mouths at distances of 6.4, 8.6, and 8.8 A (+/- 2 A) from the oxygen atoms of exposed carbonyl groups of D-Leu 12, 14, and 10, respectively. The bounded Mn2+ retains its hydrate shell, the size of which (approximately 6 A) exceeds the inner pore diameter (approximately 4 A). That makes the gramicidin A channel impermeable for divalent cations.     

Voltage-dependent formation of gramicidin channels in lipid bilayers.

The formation kinetics of gramicidin A channels in lipid bilayer membranes has been characterized as a function of voltage for different solution conditions and membrane composition. The frequency of channel events was measured during the application of voltage ramps and counted in given intervals, a procedure that eliminated the effects of drift in gramicidin concentration. The formation rate was found to increase strongly with voltages up to approximately 50 mV and then to level off slightly. The shape of the voltage dependence was independent of lipid solvent and ramp speed but differed for different ions and different solution concentrations. This suggested an ion occupancy effect on the formation rate that was further supported by the fact that the minimum of the formation rate was shifted toward the equilibrium potential in asymmetric solution concentrations. The effects are explained in terms of a model that contains two contributions to the voltage dependence, a voltage-dependent ion binding to the monomers and a polarization of monomers by the applied electric field and by the occupied ions. The theory is found to give a good fit to experimental data.     

Cation binding in Na,K-ATPase, investigated by 205Tl solid-state NMR spectroscopy

Cation binding to Na,K-ATPase is characterized in native membranes at room temperature by solid-state NMR spectroscopy using the K(+) congener (205)Tl. It has been demonstrated that the signals from occluded Tl(+) and nonspecifically bound Tl(+) can be detected and distinguished by NMR. Effects of dipole-dipole coupling between (1)H and (205)Tl in the occlusion sites show that the ions are rigidly bound, rather than just occluded. Furthermore, a low chemical shift suggests occlusion site geometries with a relatively small contribution from carboxylate and hydroxyl groups. Nonspecific binding of Tl(+) is characterized by rapid chemical exchange, in agreement with the observed low binding affinity.     

Use of weak acids to determine the bulk diffusion limitation of H+ ion conductance through the gramicidin channel.

The addition of 2 M formic acid at pH 3.75 increased the single channel H+ ion conductance of gramicidin channels 12-fold at 200 mV. Other weak acids (acetic, lactic, oxalic) produce a similar, but smaller increase. Formic acid (and other weak acids) also blocks the K+ conductance at pH 3.75, but not at pH 6.0 when the anion form predominates. This increased H+ conductance and K+ block can be explained by formic acid (HF) binding to the mouth of the gramicidin channel (Km = 1 M) and providing a source of H+ ions. A kinetic model is derived, based on the equilibrium binding of formic acid to the channel mouth, that quantitatively predicts the conductance for different mixtures of H+, K+, and formic acid. The binding of the neutral formic acid to the mouth of the gramicidin channel is directly supported by the observation that a neutral molecule with a similar structure, formamide (and malonamide and acrylamide), blocks the K+ conductance at pH 6.0. The H+ conductance in the presence of formic acid provides a lower bound for the intrinsic conductance of the gramicidin channel when there is no diffusion limitation at the channel mouth. The 12-fold increase in conductance produced by formic acid suggests that greater than 90% of the total resistance to H+ results from diffusion limitation in the bulk solution.     

Monitoring active site alterations upon mutation of yeast pyruvate kinase using 205Tl+ NMR

The interaction of the monovalent cation with wild type (WT) yeast pyruvate kinase (YPK) and with the T298S, T298C, and T298A mutants was investigated by 205Tl+ NMR to monitor possible structural alterations at the active site by Thr-298 mutation. TlNO3 activates WT YPK with a kcat value similar to that obtained with KCl and an apparent Ka of 0.96 +/- 0.07 mm in the presence of Mn2+ and fructose 1,6-bisphosphate. With the three mutants, Tl+ is a better activator than is K+ based on kcat values. Tl+ activation and inhibition of YPK is affected by mutation of the active site Thr-298. The effect of Mn2+ on the 1/T value of 205Tl+1 in the presence of the WT and mutant YPK complexes was determined at 173 MHz (300 MHz, 1H) and 346 MHz (600 MHz, 1H). For each complex studied, 1/pT2p > 1/pT1p and 1/pT1p is frequency-dependent suggesting fast exchange conditions. The values of 1/pT1p differ for each mutant. A correlation time of 0.65 +/- 0.35 ns was estimated for the Mn2+-205Tl+ interaction. The Tl+-Mn2+ distances at the active site of YPK were calculated from the paramagnetic contribution of Mn2+ to 1/T1M of YPK-bound 205Tl+. The calculated Tl+-Mn2+ distance for the Thr-298 mutants is decreased by about 1 A from 6.0 +/- 0.2 A observed with WT. The results suggest conformational alterations at the active site of YPK where phosphoryl transfer occurs upon mutation of Thr-298. These conformational changes may, in part, explain the alteration in kcat and kcat/Km,PEP observed with the Thr-298 mutants.     

TI-205 nuclear magnetic resonance determination of the thermodynamic parameters for the binding of monovalent cations to gramicidins A and C.

Thermodynamic parameters for the binding of the monovalent cations, Li+, Na+, K+, Rb+, Cs+, NH4+, TI+, and Ag+, to gramicidin A and for the binding of TI+ to gramicidin C, incorporated into lysophosphatidylcholine, have been determined using a combination of TI-205 nuclear magnetic resonance spectroscopy and competition binding. The thermodynamic parameters, enthalpy and entropy, are discussed in terms of a process involving the transfer of cations from an aqueous to amide environment.     

The structure,cation binding,transport, and conductance of Gly15-gramicidin A incorporated into SDS micelles and PC/PG vesicles

To further investigate the effect of single amino acid substitution on the structure and function of the gramicidin channel, an analogue of gramicidin A (GA) has been synthesized in which Trp(15) is replaced by Gly in the critical aqueous interface and cation binding region. The structure of Gly(15)-GA incorporated into SDS micelles has been determined using a combination of 2D-NMR spectroscopy and molecular modeling. Like the parent GA, Gly(15)-GA forms a dimeric channel composed of two single-stranded, right-handed beta(6.3)-helices joined by hydrogen bonds between their N-termini. The replacement of Trp(15) by Gly does not have a significant effect on backbone structure or side chain conformations with the exception of Trp(11) in which the indole ring is rotated away from the channel axis. Measurement of the equilibrium binding constants and Delta G for the binding of monovalent cations to GA and Gly(15)-GA channels incorporated into PC vesicles using (205)Tl NMR spectroscopy shows that monovalent cations bind much more weakly to the Gly(15)-GA channel entrance than to GA channels. Utilizing the magnetization inversion transfer NMR technique, the transport of Na(+) ions through GA and Gly(15)-GA channels incorporated into PC/PG vesicles has been investigated. The Gly(15) substitution produces an increase in the activation enthalpy of transport and thus a significant decrease in the transport rate of the Na(+) ion is observed. The single-channel appearances show that the conducting channels have a single, well-defined structure. Consistent with the NMR results, the single-channel conductances are reduced by 30% and the lifetimes by 70%. It is concluded that the decrease in cation binding, transport, and conductance in Gly(15)-GA results from the removal of the Trp(15) dipole and, to a lesser extent, the change in orientation of Trp(11).     

Analysis of the binding of Ca2+ to gramicidin A in ethanol in terms of the dimer-monomer conformational equilibrium

This study reports the first direct observation of the binding of Ca2+ to gramicidin A in ethanol, analysed in terms of the polypeptide dimer-monomer conformational equilibrium. High performance size-exclusion chromatography has been successfully used to elucidate the binding mechanism and to determine the rate constants as well as the stoichiometry of the processes involved. In addition, fluorescence intensity and anisotropy measurements have revealed a dependence of the number of accessible Ca2+-binding sites on the peptide concentration.