Experimental and theoretical studies on TI+ interactions with the cation-selective channel of the sarcoplasmic reticulum

Summary This paper presents an experimental study and a theoretical interpretation of the effects of thallous ion on the electrical properties of the cation-selective channel of the sarcoplasmic reticulum (SR channel). The properties of this channel in solutions which do not contain thallous ion are consistent with the predictions of Läuger's theory for singly occupied pores (P. Läuger, 1973,Biochim. Biophys. Acta 311:423–441). However, this theory does not account for SR channel properties in mixtures containing thallous ion. SR channel conductance is less than predicted in mixed salt solutions of thallium with either potassium or ammonium (J. Fox, 1983,Biochim. Biophys. Acta 736:241–245), yet is greater than expected in mixtures of lithium and thallium. In a simple single-ion pore, the ratio of the products of the single-salt binding constants and maximum conductances is equal to the permeability ratio calculated from zero-current potential experiments under near equilibrium conditions. This is not found for the SR channel when thallous ion is present. SR channel properties in the presence of thallous ion can, however, be explained by a model which postulates the existence of two external modulatory sites on the channel, without implying double-occupancy in the permeation pathway. When thallous ion is bound to a modulatory site the maximum conductance of the channel to all permeating ions is altered (thallous included). Two other models (a three-barrier, two-internal-site pore which allows multiple occupancy, and a pore with fluctuating barriers) are discussed, but are found to be unable to fit our conductance data at different concentrations.     

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.     

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.     

Thallous ion permeation through the cation-selective channel of the sarcoplasmic reticulum. Anomalous mole fraction dependence

The thallous ion was found to permeate the cation-selective channel of rabbit sarcoplasmic reticulum and to block current through this channel when present in mixtures with other permeant ions. Channel conductance in pure thallium acetate saturates with increasing concentration, with a maximum limiting conductance of 60 pS. The conductance ratio GK/GTl at 1 M is 3.7, while the permeability ratio is near 0.4 over the concentration range 0.01 to 1 M. Thallium blockade in mixtures can be described by the equation of Neher (Neher, E. (1975) Biochim. Biophys. Acta 401, 540-544).     

Chromatography of carboxylic proteinases on sorbents containing the 2,4-dinitrophenyl group. Role of ionic interactions]

The chromatography of porcine pepsin on biospecific sorbents (Sepharose-4B-epsilon-DNP-aminocapronylhydrazide and Sepharose-4B-N-DNP-N'-acetylhexamethylenediamine) was studied. The sorbents in question differ from the previously used hydrophobic sorbent Sepharose-4B-DNP-hexamethylenediamine by the lack of strongly basic groups in the site of the ligand binding to the polymeric matrix. No qualitative differences in the pepsin chromatography on the three sorbents were observed. Presumably the decrease of the pepsin binding to the sorbents, containing the dinitrophenyl group, at pH values above the isoelectric point may be due to the effects of the salt on the binding site in the enzyme molecule rather than to the screening of the positive charges of the sorbent by chlorine ions. A commercial preparation of pepsin was purified 2-fold on the sorbent Sepharose-4B-epsilon-DNP-animocapronylhydrazide. The synthesis of sorbents is described.     

Dynamic ion-ion and water-ion interactions in ion channels.

The dynamic interactions among ions and water molecules in ion channels are treated based on an assumption that ions at binding sites can be knocked off by both transient entering ions and local water molecules. The theory, when applied to a single-site model K+ channel, provides solutions for super- and subsaturations, flux-ratio exponent (n') greater than 1, osmotic streaming current, activity-dependent reversal potentials, and anomalous mole-fraction behavior. The analysis predicts that: (a) the saturation may but, in general, does not follow the Michaelis-Menten relation; (b) streaming current results from imbalanced water-ion knock-off interactions; (c) n' greater than 1 even for single-site channels, but it is unlikely to exceed 1.4 unless the pore is occupied by one or more ion(s); (d) in the calculation involving two permeant ion species with similar radii, the heavier ions show higher affinity; the ion-ion knock-off dissociation from the site is more effective when two interacting ions are identical. Therefore, the "multi-ion behaviors" found in most ion channels are the consequences of dynamic ion-ion and water-ion interactions. The presence of these interactions does not require two or more binding sites in channels.     

The interaction of various lanthanide ions and some anions with phosphatidylcholine vesicle membranes. A 31P NMR study of the surface potential effects

The interaction of various lanthanide ions with vesicles of phosphatidylcholine from egg yolk has been followed by 31P NMR at 30 degrees C. From known magnetic properties of these ions, separation of the paramagnetic shift into a pure contact and a pseudo-contact part was carried out. Binding curves for the contact contribution (F curves) were obtained from vesicles in solutions of sodium salts with monovalent anions over a wide concentration range. These curves should be insensitive to any conformational effects due to ion binding. Indication of a conformational change in the lipid head group at low ion binding was obtained by studying the ratio between the contact and the pseudo-contact contributions. Besides the adsorption of lanthanide ions, specific anion binding to the surface was introduced to account for the enhanced chemical shifts (Cl- < Br- < NO3-). The results were analyzed in terms of the theory for the diffuse double layer (Gouy-Chapman-Grahame) with equilibrium conditions for the adsorbing cations and anions. Simulations of the titration curves furnished parameters for the ion-lipid interactions. The synergism between the cations and anions follows from the potential effects. Comparison of results with lanthanide ions and Ca2+ indicates that the anion adsorption probably depends on the nature of the adsorbed cation. Lanthanide ion binding to L-glycerophosphorylcholine is not influenced by sodium salts. The binding constant for this complex is weaker than with phosphatidylcholine. The chemical shifts for the lanthanide ion complexes with these two phosphorus compounds seem to be about the same.     

Quantitative analysis of monovalent counterion binding to random-sequence, double-stranded DNA using the replacement ion method

A variation of affinity capillary electrophoresis, called the replacement ion (RI) method, has been developed to measure the binding of monovalent cations to random sequence, double-stranded (ds) DNA. In this method, the ionic strength is kept constant by gradually replacing a non-binding ion in the solution with a binding ion and measuring the mobility of binding and non-binding analytes as a function of binding ion concentration. The method was validated by measuring the binding of Li+ ions to adenosine nucleotides; the apparent dissociation constants obtained by the RI method are comparable to literature values obtained by other methods. The binding of Tris+, NH4+, Li+, Na+, and K+ to dsDNA was then investigated. The apparent dissociation constants observed for counterion binding to a random-sequence 26-base pair (bp) oligomer ranged from 71 mM for Tris+ to 173 mM for Na+ and K+. Hence, positively charged Tris buffer ions will compete with other monovalent cations in Tris-buffered solutions. The bound cations identified in this study may correspond to the strongly correlated, tightly bound ions recently postulated to exist as a class of ions near the surface of dsDNA (Tan, Z.-J., and Chen, S.-J. (2006) Biophys. J. 91, 518-536). Monovalent cation binding to random-sequence dsDNA would be expected to occur in addition to any site-specific binding of cations to A-tracts or other DNA sequence motifs. Single-stranded DNA oligomers do not bind the five tested cations under the conditions investigated here.     

Nonequivalence of the metal binding sites in vanadyl-labeled human serum transferrin.

Vanadyl ion, VO(IV), has been used as an electron paramagnetic resonance (EPR) spin label to study the metal-binding properties of human serum transferrin in the presence of bicarbonate. Iron-saturated transferrin does not bind the vanadyl ion. Room temperature titrations of apotransferrin with VO(IV) as monitored by EPR indicate the extent of binding to be pH dependent, with a full 0.2 VO(IV) ions per transferrin molecule bound at pH 7.5 and 9, but only about 1.2 VO(IV) ions bound at pH 6. The EPR spectra of frozen solutions with or without 0.1 M NaCUO4 at 77 K show that there are two spectroscopically nonequivalent binding sites (A and B) with a slight difference in binding constants. One site (A site) exhibits essentially constant binding capacity in the pH range 6-9, but the other (B site) becomes less avialable as the pH is reduced below 7. Results with mixed Fe(III)-VO(IV) transferrin complexes suggest that iron shows a slight tendency to bind at the B site over the A site pH 7.5 and 9.0. Only the B site in both vanadyl and iron transferrins is perturbed by the presence of perchlorate.     

Identification of mixed di-cation forms of G-quadruplex in solution

Multinuclear NMR study has demonstrated that G-quadruplex adopted by d(G3T4G4) exhibits two cation binding sites between three of its G-quartets. Titration of tighter binding K+ ions into the solution of d(G3T4G4)2 folded in the presence of 15NH4+ ions uncovered a mixed mono-K+-mono-15NH4+ form that represents intermediate in the conversion of di-15NH4+ into di-K+ form. Analogously, 15NH4+ ions were found to replace Na+ ions inside d(G3T4G4)2 quadruplex. The preference of 15NH4+ over Na+ ions for the two binding sites is considerably smaller than the preference of K+ over 15NH4+ ions. The two cation binding sites within the G-quadruplex core differ to such a degree that 15NH4+ ions bound to the site, which is closer to the edge-type loop, are always replaced first during titration by K+ ions. The second binding site is not taken up by K+ ion until K+ ion already resides at the first binding site. Quantitative analysis of concentrations of the three di-cation forms, which are in slow exchange on the NMR time scale, at 12 K+ ion concentrations afforded equilibrium binding constants. K+ ion binding to sites U and L within d(G3T4G4)2 is more favorable with respect to 15NH4+ ions by Gibbs free energies of approximately -24 and -18 kJ mol(-1) which includes differences in cation dehydration energies, respectively.     

Pepsin as a catalyst of peptide synthesis. Enzyme co-precipitation with emerging peptide products.

Pepsin successfully catalyzed the synthesis of several peptide derivatives from N-protected di- or tripeptides and amino acid or peptide esters or p-nitroanilides in dimethylformamide-water solutions at pH 4.6. An optimal substrates:pepsin ratio depended on the structure of starting peptides, especially their fit to the substrate binding sites of the enzyme. For hexapeptide Z-Ala-Ala-Phe-Leu-Ala-Ala-OCH3 formation, an equilibrium yield was attained at 1:3.10(5) enzyme-substrates ratio that indicated high efficiency of pepsin in synthesis reactions. In the course of the equilibrium peptide synthesis, pepsin gradually disappeared from the liquid phase due to its entrapment within a gel, formed by the hexapeptide product, while retaining its activity. The inclusion into the precipitate was not specific for pepsin, so far as inert proteins, lysozyme, ribonuclease A and carbonic anhydrase, when added to the reaction mixture, became also co-precipitated with the hexapeptide formed. It appears that co-precipitation of pepsin, an important factor limiting the enzyme efficiency, might be operative as well for other proteinases used to catalyze peptide synthesis.     

Anomalous mole fraction effect, electrostatics, and binding in ionic channels.

Ionic channels bathed in mixed solutions of two permeant electrolytes often conduct less current than channels bathed in pure solutions of either. For many years, this anomalous mole fraction effect (AMFE) has been thought to occur only in single-file pores containing two or more ions at a time. Most thinking about channels incorporates this view. We show here that the AMFE arises naturally, as an electrostatic consequence of localized ion specific binding, if the average current through a channel is described by a theory (Poisson-Nernst-Planck, PNP) that computes the average electric field from the average concentration of charges in and near the channel. The theory contains only those ion-ion interactions mediated by the mean field, and it does not enforce single filing. The AMFE is predicted by PNP over a wide range of mean concentrations of ions in the channel; for example, it is predicted when (on the average) less, or much less, than one ion is found in the channel's pore. In this treatment, the AMFE arises, in large measure, from a depletion layer produced near a region of ion-specific binding. The small excess concentration of ions in the binding region repels all nearby ions of like charge, thereby creating a depletion layer. The overall conductance of the channel arises in effect from resistors in series, one from the binding region, one from the depletion zone, and one from the unbinding region. The highest value resistor (which occurs in the depletion zone) limits the overall series conductance. Here the AMFE is not the result of single filing or multiple occupancy, and so previous views of permeation need to be revised: the presence of an AMFE does not imply that ions permeate single file through a multiply occupied pore.     

Metal ion complexes of apoferritin. Evidence for initial binding in the hydrophilic channels

Metal binding to the iron storage protein apoferritin is the first step in the process by which iron accumulates within the protein shell. In the present study, the stoichiometry of metal binding to apoferritin in solution has been examined using the probe ions Mn(II), VO(IV), and Cd(II) in conjunction with EPR spectroscopic and cadmium ion selective electrode measurements. Binding studies were carried out with the individual ions, in competition with one another, and in competition with Fe(II), Fe(III), and Tb(III). All three probe ions show binding stoichiometries near 0.3 and 0.7 metal ion per subunit, close to the theoretically predicted values of 0.33 and 0.67 for the binding of one and two metal ions, respectively, per three subunits. These results in conjunction with other data are consistent with the binding of one, and possibly two, metal ions within each of the eight hydrophilic channels which are located on 3-fold axes leading to the interior of the protein. Pairs of cadmium binding sites have been located in these channels by x-ray crystallography (Rice, D. W., Ford, G. C., White, J. L., Smith, J. M. A., and Harrison, P. M. (1983) Adv. Inorg. Biochem. 5, 39-49). The possibility that some metal binding occurs elsewhere on the protein is not precluded by the present data, however. In competition experiments between various metal ions, approximately 0.3 metal ion per subunit is readily displaced implying common binding sites in the channels for all of them. The stoichiometry of Mn(II) displacement by Fe(II) is less clear. Oxidation of Fe(II) to Fe(III) by molecular oxygen in the presence of Mn(II) regenerates some Mn(II) binding on the protein, suggesting migration of iron(III) to other protein sites, or perhaps to core.     

Analysis of interaction between chaperonin GroEL and its substrate using fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) provides information about translational diffusion properties of fluorescent molecules in tiny detection volume and allows the analysis of binding processes of biomolecules in homogeneous solution. In this study, FCS was used to measure equilibrium binding constants of disulfide-reduced apo-alpha-lactalbumin (rLA), denatured pepsin, and apo-cytochrome c (apo-cyt c) bound by chaperonin GroEL at different salt concentrations. The results indicate that apo-cyt-c has a much stronger affinity to GroEL than denatured pepsin and rLA have. Titration experiments of GroEL to each substrate with various concentrations of four kinds of salts (K+, Na+, Ca2+, and Mg2+) show that the binding constant of denatured pepsin and rLA to GroEL depends on the salt concentration. The dependence of denatured pepsin binding to GroEL on salt concentration is much stronger than that of rLA. However, the interaction of positively charged apo-cyt c with GroEL is not affected by the salt concentration. Furthermore, the divalent cation promotes the binding of GroEL to denatured pepsin and rLA more strongly than does the monovalent cation.     

Interaction between quaternary ammonium ions in the pore of potassium channels. Evidence against an electrostatic repulsion mechanism

We have examined the interaction between internal and external ions in the pore of potassium channels. We found that external tetraethylammonium was able to antagonize block of Shaker channels by internal TEA when the external and internal solutions contained K(+) ions. This antagonism was absent in solutions with Rb(+) as the only permeant ion. An externally applied trivalent TEA analogue, gallamine, was less effective than the monovalent TEA in inhibiting block by internal TEA. In addition, block by external TEA was little affected by changes in the concentration of internal K(+) ions, but was increased by the presence of internal Na(+) ions in the pore. These results demonstrate that external and internal TEA ions, likely located at opposite ends of the pore selectivity filter, do not experience a mutual electrostatic repulsion. We found that these results can be simulated by a simple 4-barrier-3-site permeation model in which ions compete for available binding sites without long-range electrostatic interactions.     

The influence of Al3+ ion on porcine pepsin activity in vitro

The in vitro effect of Al³⁺ ions in the concentration range 1.7·10^{? 6} M–8.7·10^{? 3} M on pepsin activity at pH 2, via kinetic parameters and its electrophoretic mobility was evaluated. Kinetic study demonstrated the existence of an activation effect of Al³⁺ at pH 2 on pepsin molecule. Kinetic analysis with respect to concentrations of haemoglobin showed that Al³⁺ ions increase the maximal velocity (V_max) and k_cat values rather than apparent affinity for substrate (K_S) implying the non-competitive nature of activation which indicated that aluminium was a non-essential activator of partial non-competitive type. The values of the equilibrium constants K_S and K_mA for dissociation of corresponding complexes were evaluated as 0.904 ± 0.083 mM and 8.56 ± 0.51 μM, respectively. Dissociation constant K_A, of activator from enzyme-activator complex calculated via kinetic and direct measurement of Al³⁺ binding data, as well as activation constant A₅₀, the activator concentration that gives a rate equal to half at a saturating concentration of activator, were found to be 8.82 ± 0.90 μM, 8.39 ± 0.76 μM, and 8.05 ± 0.48 μM respectively. Native PAGE electrophoresis shows the decrease in electrophoretic mobility of pepsin and confirms modification of the electric charge and conformational changes of pepsin caused by bound Al³⁺ on the pepsin molecule. Al³⁺ induced conformational changes of pepsin were verified by UV-VIS and IR spectra. Moreover, the absence of conformational changes in the haemoglobin molecule in the presence of Al³⁺ ions confirms that the obtained activation is a consequence of conformational changes caused only in the pepsin molecule.     

Dielectric properties of polyelectrolytes. II. A theory of dielectric increment due to ion fluctuation by a matrix method

The mean square of dipole moment of a linear macromolecule which is responsible for dielectric increment of aqueous polyelectrolyte solutions is calculated by means of a matrix method in which ion binding at discrete sites and the nearest-neighbor interaction are taken into account. On the basis of the relationship between polarization of poly-ion and fluctuation of bound counterions the present theory indicates that the loosely bound ions result in larger increment and otherwise smaller increment. Also, the theory predicts that the dielectric increment has a maximum at an intermediate monovalent–divalent ion ratio when both species coexist. These results are consistent with experiments on polyacrylic acid neutralized with NaOH and Ca(OH)₂. At large contents of divalent ions the effect of chelation is also discussed.     

Isothermal titration calorimetric procedure to determine protein-metal ion binding parameters in the presence of excess metal ion or chelator

Determination of binding parameters for metal ion binding to proteins usually requires preceding steps to remove protein-bound metal ions. Removal of bound metal ions from protein is often associated with decreased stability and inactivation. We present two simple isothermal titration calorimetric procedures that eliminate separate metal ion removal steps and directly monitor the exchange of metal ions between buffer, protein, and chelator. The concept is to add either excess chelator or metal ion to the protein under investigation and subsequently titrate with metal ion or chelator, respectively. It is thereby possible in the same experimental trial to obtain both chelator-metal ion and protein-metal ion binding parameters due to the different thermodynamic "fingerprints" of chelator and protein. The binding models and regression routines necessary to analyze the corresponding binding isotherms have been constructed. Verifications of the models have been done by titrations of mixtures of calcium chelators (BAPTA, HEDTA, and EGTA) and calcium ions and they were both able to account satisfactorily for the observed binding isotherms. Therefore, it was possible to determine stoichiometric and thermodynamic binding parameters. In addition, the concept has been tested on a recombinant alpha-amylase from Bacillus halmapalus where it proved to be a consistent procedure to obtain calcium binding parameters.     

Study on the interaction of β‐carotene and astaxanthin with trypsin and pepsin by spectroscopic techniques

β‐Carotene and astaxanthin are two carotenoids with powerful antioxidant properties, but the binding mechanisms of β‐carotene/astaxanthin to proteases remain unclear. In this study, the interaction of these two carotenoids with trypsin and pepsin was investigated using steady‐state and time‐resolved fluorescence measurements, synchronous fluorescence spectroscopy, UV–vis absorption spectroscopy and circular dichroism (CD) spectroscopy. The experimental results indicated that the quenching mechanisms of trypsin/pepsin by the two carotenoids are static processes. The binding constants of trypsin and pepsin with these two carotenoids are in the following order: astaxanthin–trypsin > astaxanthin–pepsin > β‐carotene–trypsin > β‐carotene–pepsin, respectively. Thermodynamic investigations revealed that the interaction between the two carotenoids and trypsin/pepsin is synergistically driven by enthalpy and entropy, and hydrophobic forces and electrostatic attraction have a significant role in the reactions. In addition, as shown by synchronous fluorescence spectroscopy, UV–vis absorption spectroscopy and CD, the two carotenoids may induce conformational and microenvironmental changes in trypsin/pepsin. The study provides an accurate and full basic data for clarifying the binding mechanisms of the two carotenoids with trypsin/pepsin and is helpful in understanding their effect on protein function and their biological activity in vivo. Copyright © 2015 John Wiley & Sons, Ltd.     

A strategy for high-throughput screening of ligands suitable for molecular imprinting of proteins

For facilitating the identification of appropriate functionalities that may serve as a binding motif of functional monomers, a selection strategy based on high-throughput screening of the binding properties of readily available sorbent materials has been developed. Thereby, the affinity of such ligands to the protein of interest may be rapidly determined. From these studies, it is anticipated that ligand functionalities will be derived, which may lead to advanced selection and design of dedicated functional monomers suitable for decorating the surface of a scavenger material. Thus, specific binding of the target protein of interest should be enabled even in complex solutions such as e.g., biotechnologically relevant cell lysates. In the present contribution, an automated screening method for studying ligand interactions of selected sorbent materials with pepsin - a protein of the protease family - was developed. Aqueous buffer solutions containing pepsin at known constant concentration were pipetted through an array of miniaturized chromatographic solid phase extraction (SPE) columns containing a variety of sorbent materials, and the eluted solutions were analyzed by UV/vis spectroscopy. The established screening protocol was validated against resin materials of known interaction with pepsin. Finally, the developed screening strategy was adapted for a robot system enabling high-throughput screening for a wide variety of sorbent materials and ligand functionalities in a fully automated approach. The obtained results clearly indicate that the established screening routine provides valuable data for characterizing resin-immobilized ligands, and their affinity toward pepsin.