Conformational studies of A23187 with mono-, di- and trivalent metal ions by circular dichroism spectroscopy

CD studies carried out on A23187 indicate a solvent-dependent conformation for the free acid. Alkali metal ions were found to bind to the ionophore weakly. Divalent metal ions such as Mg2+, Ca2+, Sr2+, Ba2+ and Co2+ and trivalent lanthanide metal ions like La3+ were found to form predominantly 2:1 (ionophore-metal ion) complexes at low concentrations of metal ions, but both 2:1 and 1:1 complexes were formed with increasing salt concentration. Mg2+ and Co2+ exhibit similar CD behaviour that differs from that observed for the other divalent and lanthanide metal ions. The structure of 2:1 complexes involves two ligand molecules coordinated to the metal ion through the carboxylate oxygen, benzoxazole nitrogen and keto-pyrrole oxygen from each ligand molecule along with one or more solvent molecules. Values of the binding constant were determined for 2:1 complexes of the ionophore with divalent and lanthanide metal ions.     

A merged experimental and theoretical conformational study on alkaline-earth complexes with lariat ethers derived from 4,13-diaza-18-crown-6

Herein, we report the synthesis and structural characterization of alkaline-earth complexes with the bibracchial lariat ethers N,N′-bis(2-aminobenzyl)-4,13-diaza-18-crown-6 (L²) and N,N′-bis(benzimidazol-2ylmethyl)-4,13-diaza-18-crown-6 (L⁴). The X-ray crystal structures of the Ca(II) and Sr(II) complexes of L² show the pendant arms of the ligand disposed on opposite sides of the macrocyclic mean plane, which results in an anti conformation in the solid state. A similar anti conformation is also observed for the Mg(II) complex of L⁴, whereas the Ca(II), Sr(II) and Ba(II) complexes of L⁴ adopt a syn conformation in the solid state, with the two pendant arms pointing at the same side of the crown moiety. However, a different behavior is observed in solution. Indeed, ¹H and ¹³C NMR spectroscopy, in combination with density functional theory (DFT) calculations performed at the B3LYP level, suggests that the [M(L²)]²⁺ and [M(L⁴)]²⁺ (M = Ca, Sr or Ba) complexes exist in solution as a mixture of syn and anti isomers involved in a dynamic equilibrium. Our results also show that the relative abundance of the syn conformation increases as the ionic radius of the metal ion increases and, furthermore, for a given metal ion the proportion of syn isomer is always higher for L⁴ complexes than for L² ones.     

Calix[4]arene based molecular sensors with pyrene as fluoregenic unit: Effect of solvent in ion selectivity and colorimetric detection of fluoride

Fluorescent molecular sensors having excimer emission property have been designed and synthesised incorporating calix[4]arene derivatives in cone and 1,3-alternate conformation as ionophore and two pyrene moieties at close proximity as fluorophore. They exhibit strong excimer emission around 515 nm, which is used to monitor interaction of metal ions with the ionophores. Ion-binding study of these fluoroionophore has been investigated in acetonitrile-chloroform and THF-H₂O with a wide range of cations and anions and the recognition process is monitored by luminescence, UV-Vis and ¹H NMR (for F⁻) spectral changes. The present study demonstrated profound influence of solvent in ion selectivity, in acetonitrile-chloroform they formed complexes with Hg²⁺, Pb²⁺, Cu²⁺ and Ni²⁺, whereas in THF-H₂O they exhibit selectivity only for Cu²⁺. In the case of anions, selectivity for only F⁻ with color change is observed. Composition of the complexes formed was determined from mass spectrometry and the binding constants were determined from fluorescence titration data. The reasons for formation of excimer emission, quenching of it in presence of certain metal ions, role of solvent in selectivity and energy/electron transfer process involved in the ion-recognition event have been discussed on the basis of experimental data.     

Metal Ion Binding and Conformational Transitions in Concanavalin A: A Structure-Function Study

Abstract

The affinity of the lectin Concanavalin A (Con A) for saccharides, and its requirement for metal ions such as Mn²⁺ and Ca²⁺, have been known for about 50 years. However the relationship between metal ion binding and the saccharide binding activity of Con A has only recently been examined in detail. Brown et al. (Biochemistry 16, 3883 (1977)) showed that Con A exists as a mixture of two conformational states: a “locked” form and an “unlocked” form. The unlocked form of the protein weakly binds metal ions and saccharide, and is the predominate conformation of demetallized Con A (apo-Con A) at equilibrium. The locked form binds two metal ions per monomer with the resulting complex(es) possessing full saccharide binding activity. Brown and coworkers measured the kinetics of the transition of the unlocked form to the fully metallized locked conformation containing Mn²⁺and Ca²⁺. They also demonstrated that Mn²⁺ alone could form a locked ternary complex with Con A, and that rapid removal of the ions resulted in a metastable form of apo-Con A in the locked conformation which slowly (hours at 25°C) reverted back to (predominantly) the unlocked conformation. The ability to form either conformation in the absence or presence of metal ions has thus allowed us to explore the relationship between metal ion binding and conformational transitions in Con A as determinants of the saccharide binding activity of the lectin.

Based on the kinetics of the transition of unlocked apo-Con A to fully metallized locked Con A, and X-ray crystallographic data, it appears that the transition between the two conformations of Con A involves a cis-trans isomerization of an Ala-Asp peptide bond in the backbone of the protein, near one of the two metal ion binding sites. The relatively large activation energy for the transition (～ 22 kcal M^?1) results in relatively slow interconversions between the conformations (from minutes to days), whereas the equilibria with metal ions and saccharide are rapid. Thus, many metastable complexes can be formed and a variety of transition pathways between the two conformations studied.

We have identified and characterized binary, ternary, and quaternary complexes of both conformations of Con A containing Mn²⁺ and saccharide, and have determined both metalion and saccharide dissociation constants for all of them, as well as equilibrium and kinetic values for the conformational transitions between them. The main finding is that saccharide binds very weakly (K_d～2 M) to unlocked apo-Con A and very tightly to the locked ternary Mn²⁺-Con A complex (K_d～ 10^?4 M). Saccharide binding increases along the various pathways connecting these two species in a nonadditive fashion. Thus, both conformation and metal ion binding determine the saccharide affinity of each complex, although the specificity of saccharide binding of the various species is maintained throughout.     

The interaction of analogues of the antimicrobial lipopeptide, iturin A2, with alkali metal ions

Electrospray mass spectrometry was employed as a tool in this first study on the molecular interaction between the alkali metal ions and antifungal lipopeptide iturin A, and some analogues. Cationisation by sodium and signal intensity of lipopeptide species depended on sodium concentration, but was independent of sample solvent, carrier solvent polarity and sample pH between 4 and 11. 8-Beta, a linear analogue of iturin A2 (8-Beta; beta-aminotetradecanoyl-NYNQPNS), and its shorter linear lipopeptide analogues, associated either one or two alkali metal cations, while the N-->C cyclic peptides associated with only one cation. The chirality of the beta-NC14 residue had a limited influence on the cationisation. It was observed that 8-Beta contained at least four interaction sites for a cation of which two, the C-terminal carboxylate and the side-chain of tyrosine, can take part in ionic interaction with a cation. It is proposed that the remaining two interaction centres of alkali metal ions are within the two type II beta-turns found in conformation of natural iturin A. This was corroborated by the diminished capacity of the shorter peptides, in which one of the beta-turns was eliminated to bind a second larger cation. All the lipopeptides showed the same order of alkali metal ion selectivity: Na+ > K+ > Rb+. These results indicated a size limitation in the interaction cavity or cavities. The absence of, or observation of only low abundance, di-cationised complexes of cyclic peptides the indicated association of the cation in the interior of the peptide ring. It is thus hypothesised that alkali metal ions can bind in one of the two beta-turns in the natural iturin A molecule.     

A model for metalloprotein-catalysed ADP, ATP transport in mitochondria

We propose a hypothetical model for the transmembrane exchange reaction catalysed by the mitochondrial adenine nucleotide carrier protein, which basically consists of an alternating reorientation of a transitory carrier—metal—nucleotide complex. The key features of the model are: the participation of an intrinsic divalent metal ion in the course of transport catalysis; the different stability constants of protonated and deprotonated nucleotide—metal complexes; the exposure and retraction of strategic arginyl residues; the alternating reorientation of the active center involving a change from the cytosolic conformation (C_c) to the matrix conformation (C_m).     

The transport of Na+ and K+ ions through phospholipid bilayers mediated by the antibiotics salinomycin and narasin studied by 23Na- and 39K-NMR spectroscopy

Addition of the ionophoric antibiotics salinomycin or narasin to preparations of large unilamellar vesicles made from egg yolk phosphatidylcholine in sodium or potassium chloride solutions gives rise to dynamic effects in the 23Na- and 39K-NMR spectra. The dynamic spectra arise from the ionophore-mediated transport of the metal ions through the membrane. The kinetics of the transport are followed as a function of the concentrations of ionophore and the metal ion and are compatible in all cases with a model in which one ionophore molecule transports one metal ion. For both ionophores the transport of potassium ions is appreciably faster than that of sodium and in both cases the rate-limiting step for sodium transport is dissociation of the ionophore-metal complex. Assuming dissociation to be rate limiting in all four cases it is shown that the transport rate differences between the pairs of complexes of each metal arise solely from differences in the rates of formation. The stability constants for ionophore-metal complex formation in the membrane/water interface are evaluated.     

Fluorescence study of the divalent cation-transport mechanism of ionophore A23187 in phospholipid membranes

The mechanism for transport of divalent cations across phospholipid bilayers by the ionophore A23187 was investigated. The intrinsic fluorescence of the ionophore was used in equilibrium and rapid-mixing experiments as an indicator of ionophore environment and complexation with divalent cations. The neutral (protonated) form of the ionophore binds strongly to the membrane, with a high quantum yield relative to that in the aqueous phase. The negatively charged form of the ionophore binds somewhat less strongly, with a lower quantum yield, and does not move across the membrane. Complexation of the negatively charged form with divalent cations was measured by the decrease in fluorescence. An apparent rate constant (kapp) for transport of the ionophore across the membrane was determined from the rate of fluorescence changes observed in stopped-flow rapid kinetic experiments. The variation of kapp was studied as a function of pH, temperature, ionophore concentration, membrane lipid composition, and divalent cation concentration and type. Analysis and comparison with equilibrium constants for protonation and complexation show that A23187 and its metal:ionophore complexes bind near the membrane-water interface in the lipid polar-head region. The interfacial reactions occur rapidly, compared with the transmembrane reactions, and are thus in equilibrium during transport. The transport cycle can be described as follows: a 1:1 complex is formed between the membrane bound A23187-(Am-) and the aqueous divalent cation with dissociation constant K1 approximately 4.6 x 10(-4) M. This is in equilibrium with a 1:2 (metal:ionophore) complex (K2 approximately 3.0 x 10(-4) [ionophore/lipid]) that is responsible for transporting the divalent cations across the membrane. The rate constant for translocation of the 1:2 complex is 0.1-0.3 s-1. Dissociation of the complex of the trans side and protonation occur rapidly. The rate constant for translocation of H+ . A23187- is 28 s-1. A theory is presented that is capable of reproducing the kinetic data at any calcium concentration. The cation specificity for ionophore complex transport (kapp), determined at low ionophore concentration for a series of divalent cations, was found to be proportional to the equilibrium constant for 1:1 complexation. The order of ion specificity for these processes was found to be Ca2+ greater than Mg2+ greater Sr2+ greater than Ba2+. Interactions with Na+ were not observed. Maximal values of kapp were observed for vesicles prepared from pure dimyristoyl phosphatidylcholine. Inclusion of phosphatidyl ethanolamine, phosphatidic acid, or dipalmatoyl phosphatidylcholine resulted in lower values of kapp. Calcium transport by A23187 is compared with that of X537A, and it is shown that the former is 67-fold faster. The difference in rates is due to differences in the ability of each ionophore to form a 1:2 complex from a 1:1 complex.     

Cu(II)—(lAsp)n system. Spectroscopic evidence of hexatomic chelate ring formation

The study of the Cu(II)-(L Asp)_n system using circular dichroism and potentiometric data has provided evidence indicating the formation of two complexes in a two step process. In the first (I) of these complexes, obtained at pH 4.5, two carboxyl residues are bound to the metal. This complex partially inhibits the transition from α helix to nonperiodic conformation. In the second complex (II) two peptide nitrogens and two carboxylate oxygens are bound to each Cu(II) ion forming two hexatomic chelate rings. The CD spectral pattern is then the opposite of what is obtained when a five-membered chelate ring is formed.     

Conformation of complexes of thiamin pyrophosphate with divalent cations as studied by nuclear magnetic resonance spectroscopy.

The binding of Ni-2+ and Mn-2+ to thiamin phosphate and thiamin pyrophosphate (thiamin-PP) has been compared with the binding of these ions to oxythiamin phosphate and oxythiamin pyrophosphate, analogues of thiamin in which the C-4 amino group has been replaced by an -OH group. The replacement of the NH2 group results in reduced basicity of N-1 of the pyrimidine ring of oxythiamine derivatives. The effects of pD, ligand concentration, and temperature on the binding of metal ions to N-1 have been studied by observing the metal ion-induced shifting and broadening of the C-6-H signal of these compounds. The results indicate the following: (a) the metal ion is held near N-1, resulting in a "folded" conformation, because of a favorable bonding interaction between N-1 and the metal ion rather than for general conformational reasons alone; and (b) the amount of "folded" conformation present in the different pyrophosphate complexes at neutral pH follows the order: Ni-2+-thiamin-PP greater than Mn-2+-thiamin-PP greater than Mn-2+-oxythiamin-PP and Ni-2+-oxythiamin-PP It is concluded that the strength of the metal ion-pyrimidine interaction in the "folded" conformation depends strongly both on the coordination affinity of the metal ion and on the basicity of N-1. Since the interaction of the phosphate-bound metal ion with the pyrimidine ring in the Mg-2+-thiamin-PP complex is probably weaker than the corresponding interaction in the Mn-2+-thiamin-PP complex, these results predict that the Mg-2+-thiamin-PP complex in solution, at neutral pH, exists predominantly in an "unfolded" conformation.     

Conformations of flanking bases in HIV-1 RNA DIS kissing complexes studied by molecular dynamics

Explicit solvent molecular dynamics simulations (in total almost 800 ns including locally enhanced sampling runs) were applied with different ion conditions and with two force fields (AMBER and CHARMM) to characterize typical geometries adopted by the flanking bases in the RNA kissing-loop complexes. We focus on flanking base positions in multiple x-ray and NMR structures of HIV-1 DIS kissing complexes and kissing complex from the large ribosomal subunit of Haloarcula marismortui. An initial x-ray open conformation of bulged-out bases in HIV-1 DIS complexes, affected by crystal packing, tends to convert to a closed conformation formed by consecutive stretch of four stacked purine bases. This is in agreement with those recent crystals where the packing is essentially avoided. We also observed variants of the closed conformation with three stacked bases, while nonnegligible populations of stacked geometries with bulged-in bases were detected, too. The simulation results reconcile differences in positions of the flanking bases observed in x-ray and NMR studies. Our results suggest that bulged-out geometries are somewhat more preferred, which is in accord with recent experiments showing that they may mediate tertiary contacts in biomolecular assemblies or allow binding of aminoglycoside antibiotics.     

Considerations of the estimation of plasmodesmatal frequencies

The association constants reported for the formation of metal ion : polyampholyte complexes can be in substantial error due to buffer effects. The magnitude of the error is a function of the affinity constants of the metal ion to the polyampholyte and to the buffer, the pH and the concentrations. A mathematical and graphical analysis of the depletion of the free metal ion concentration by formation of multiple metal ion: buffer complexes, MB_i, (where j ranges from 1 to n) is presented. An analysis is made of the dependence of the experimentally determined association constant for the formation of a 1:1 metal ion: polyampholyte complex in a system where a 1:1 complex of metal ion: buffer is also produced. Other sources of error considered include the formation of mixed complexes consisting of polyampholyte, metal ions and buffer components.By determining the pH shift resulting from protein: metal ion coordination in a buffer-free system it is possible to establish the extent of metal ion binding, gain insight about to the mechanism of the association process and ascertain whether certain types of structural transitions occur.     

Determination of the solution conformation of dephospho coenzyme A by nuclear-magnetic-resonance spectroscopy with lanthanide probes. A method for analysis when more than one complex species is present

The aqueous solution conformation of the 1 : 1 complex of dephospho CoA bound to a lanthanide ion has been determined by examination of the dipolar shift and induced relaxation at pH 6.4. The experimental data are shown to arise from the presence of both 1 : 1 and 1 : 2 metal : ligand complexes and a graphical method is described to divide the experimental data into information corresponding to each of the two species. The formation constants are also derived. For the 1 : 1 complex the ribose is found in a 2E conformation with the adenine base predominantly anti. A small contribution from a syn conformation is evident. The pantoinic acid fragment of the chain is folded back towards the pyrophosphate while the remainder of the chain is extended.     

A site-specific mutation within the active site of ribulose-1,5-bisphosphate carboxylase of Rhodospirillum rubrum

In vitro mutagenic techniques have generated an asp→glu substitution at residue 198 adjacent to the carbamate-divalent metal ion binding site of Rhodospirillum rubrum ribulose 1,5-bisphosphate carboxylase. A single C→A nucleotide change in the coding strand created the mutant and introduced a new EcoRI restriction site on the expression plasmid pRR2119. Although the carboxylase:oxygenase ratio remained the same, the mutant enzyme had slightly altered kinetic properties. The e.p.r. spectra of the quaternary complexes enzyme.activator carbamate.Mn²⁺.2-carboxyarabinitol 1,5-bisphosphate and enzyme.activator carbamate.Mn²⁺.4-carboxyarabinitol 1,5-bisphosphate for mutant and wild-type enzymes were different, indicating that the metal ion was in a slightly altered environment. These findings are consistent with the hypothesis that, besides the carbamate at lys 201, the carboxyl group of asp 198 contributes to the formation of the divalent metal ion binding site.     

CD and nmr studies on the interaction of lithium ion with valinomycin and gramicidin-S

The conformation of the valinomycin–lithium complex has been studied using CD and nmr techniques. The lithium ion induced significant changes in the chemical shifts of the NH and C^αH protons, as well as in the CD spectra of valinomycin. From the analysis of the lithium ion titration data, it is concluded that valinomycin forms a 1:1 type weak complex with lithium, having a stability constant of 48 L mol^?1 at 25°C. This conformation is different from the familiar valinomycin–potassium complex. The nature of the interaction at low and high concentrations of lithium ions with valinomycin (ionophore) and gramicidin-S (nonionophore) has been compared. At high salt concentrations, there was a further change in the CD and nmr spectra of valinomycin, giving a second plateau region at > 3M of the salt. In the case of gramicidin-S, no significant changes in the nmr or CD spectra were observed in the lower concentration range corresponding to where changes were observed in the case of valinomycin. However, the addition of lithium salt at concentrations greater than 3M induced changes in both the CD and nmr spectra of gramicidin-S, and the titration graph of molar ellipticity versus concentration of lithium perchlorate gave a plateau region at concentrations greater than this. These results indicate that the effects of lithium at low and high concentrations are independent of each other. The conformational transitions at very high salt concentrations (denaturation) are more likely due to solvent structural perturbations rather than to the consequences of ion binding.     

Influence of solvent and of cation size on the conformations of lasalocid A-lanthanide(III) ion complexes: circular dichroism and fluorescence studies

The interaction of lanthanide(III) nitrates (La3+ to Lu3+) with the carboxylic ionophore lasalocid A (LS) has been studied by circular dichroism (CD) and fluorescence spectroscopic techniques in acetonitrile and in methanol. Analysis of the CD data in acetonitrile has revealed the coexistence of both 1:1 (ionophore:cation) and 2:1 complexes in solution. For 1.22 A greater than ionic radius greater than 1.13 A, 1:1 complexes are preferred, and for 1.13 A greater than ionic radius greater than 1.03 A, 2:1 complexes are preferred. Induced CD bands for Ln3+ ions have been observed upon binding to LS in acetonitrile. The LS-Ln3+ complexes are less stable in methanol than in acetonitrile. CD spectral changes showed that the conformations of the complexes in methanol are different from those in acetonitrile. The complexes have rather open conformations in methanol compared to those in acetonitrile. The results underscore the importance of ionic radius, solvent environment, and ionization state of LS in determining the conformations of the ionophore-cation complexes.     

Electron paramagnetic resonance of copper ion and manganese ion complexes with the ionophore A23187.

The binding of Cu2+ and Mn2+ to the ionophore A23187 in chloroform, 90% ethanol, and sonicated phospholipid dispersions in aqueous mediums has been investigated with electron paramagnetic resonance (epr). The spectra indicated axial symmetry for the Cu2+ complexes and distorted octahedral for the Mn2+ complexes. The coordination between metal ion and its ligands is predominantly ionic in character. The stoichiometry, at the concentrations employed, was found to be 1:2 M2+/ionophore except in 90% ethanol where evidence existed for the 1:1 Cu-A23187 complex, as well. Through competition with Mn2+, the sequence of relative affinities in 90% ethanol was measured to be: Mn2+ greater than La3+ greater than Cu2+ greater than Ca2+ greater than Mg2+ greater than Sr2+. The K A of Mn-A23187 binding is greater than 10 10 M-2. In phospholipid dispersions the spectral characteristics of the Cu complex, particularly g, were observed to be a sensitive function of the hydrocarbon chain mobility. This allowed a calculation of the rotational correlation time of the complex to be made. In sonicated dipalmitoyllecithin was computed to be 10-9 sec, reflecting a local viscosity similar to that sensed by the nitroxide spin-label 2,2,6,6-tetramethylpiperidin-1-oxyl. In a (1:1) lecithin-cholesterol dispersion the complex was significantly more immobilized.     

Conformational characterisation of valinomycin complexation with barium salts—A nuclear magnetic resonance spectroscopic study

Conformations of valinomycin and its complexes with Perchlorate and thiocyanate salts of barium, in a medium polar solvent acetonitrile, were studied using nuclear magnetic resonance spectroscopic techniques. Valinomycin was shown to have a bracelet conformation in acetonitrile. With the doubly charged barium ion, the molecule, at lower concentrations, predominantly formed a 1:1 complex. At higher concentrations, however, apart from the 1:1, peptide as well as ion sandwich complexes were formed in addition to a ‘final complex’. Unlike the standard 1:1 potassium complex, where the ion was centrally located in a bracelet conformation, the 1:1 barium complex contained the barium ion at the periphery. The ‘final complex’ appeared to be an open conformation with no internal hydrogen bonds and has two bound barium ions. This complex was probably made of average of many closely related conformations that were exchanging very fast (on nuclear magnetic resonance time scale) among them. The conformation of the ‘final complex’ resembled the conformation obtained in the solid state. Unlike the Perchlorate anion, the thiocyanate anion seemed to have a definite role in stabilising the various complexes. While the conformation of the 1:1 complex indicated a mechanism of ion capture at the membrane interface, the sandwich complexes might explain the transport process by a relay mechanism.     

An examination of the binding behavior of histidine-containing peptides with immobilized metal complexes derived from the macrocyclic ligand, 1,4,7-triazacyclononane

In this study, two different experimental approaches have been employed to examine the binding behavior of histidine-containing peptides with metal ion complexes derived from the macrocyclic ligand 1,4,7-triazacyclononane (tacn). Firstly, a molecular modeling approach has been employed to derive the strain energies for test peptide sequences that have a predicted propensity to readily adopt an α-helical conformation. To this end, binuclear metal complexes were examined with peptides containing two histidine residues in different locations in a pair of peptides of the same composition but different sequence. These modeling results indicate that there are no energetic constraints for two-point binding to occur with dicopper(II) binuclear complexes when two histidine residues are appropriately placed in an α-helical conformation. Secondly, binding experiments were carried out to establish the effect of one or more histidine residues within a peptide sequence on the affinity of a peptide for these Cu(II)–tacn derived binuclear complexes when immobilized onto a chromatographic support material. The results confirm that for all chelating systems, higher affinity is achieved as the histidine number in the peptide structure increases, although the relative location of the histidine residues in these small peptides did not introduce a significant constraint to the conformation on interacting with the immobilized Cu(II) binuclear complexes.     

A General Approach to the Optimization of the Conformation of Ring Molecules with an Application to Valinomycin

Abstract

A general and efficient methodology is presented which allows molecules containing one or many rings of any size to be manipulated within energy minimization procedures. Variables describing the conformation of the molecules concerned are limited to dihedral and ring valence angles and the ring closure conditions are treated as equality constraints. An application is made to the ion transporter valinomycin and its complexes with K⁺ and Na⁺ which illustrates the possibilities of the approach and leads to results which allow a better understanding of the conformational mechanics of this important ionophore.