The integral divalent cation within the intermolecular purine*purine. pyrimidine structure: a variable determinant of the potential for and characteristics of the triple helical association.

In vitro assembly of an intermolecular purine*purine.pyrimidine triple helix requires the presence of a divalent cation. The relationships between cation coordination and triplex assembly were investigated, and we have obtained new evidence for at least three functionally distinct potential modes of divalent cation coordination. (i) The positive influence of the divalent cation on the affinity of the third strand for its specific target correlates with affinity of the cation for coordination to phosphate. (ii) Once assembled, the integrity of the triple helical structure remains dependent upon its divalent cation component. A mode of heterocyclic coordination/chelation is favorable to triplex formation by decreasing the relative tendency for efflux of integral cations from within the triple helical structure. (iii) There is also a detrimental mode of base coordination through which a divalent cation may actively antagonize triplex assembly, even in the presence of other supportive divalent cations. These results demonstrate the considerable impact of the cationic component, and suggest ways in which the triple helical association might be positively or negatively modulated.     

A unified model for the origin of DNA sequence-directed curvature

The fine structure of the DNA double helix and a number of its physical properties depend upon nucleotide sequence. This includes minor groove width, the propensity to undergo the B-form to A-form transition, sequence-directed curvature, and cation localization. Despite the multitude of studies conducted on DNA, it is still difficult to appreciate how these fundamental properties are linked to each other at the level of nucleotide sequence. We demonstrate that several sequence-dependent properties of DNA can be attributed, at least in part, to the sequence-specific localization of cations in the major and minor grooves. We also show that effects of cation localization on DNA structure are easier to understand if we divide all DNA sequences into three principal groups: A-tracts, G-tracts, and generic DNA. The A-tract group of sequences has a peculiar helical structure (i.e., B*-form) with an unusually narrow minor groove and high base-pair propeller twist. Both experimental and theoretical studies have provided evidence that the B*-form helical structure of A-tracts requires cations to be localized in the minor groove. G-tracts, on the other hand, have a propensity to undergo the B-form to A-form transition with increasing ionic strength. This property of G-tracts is directly connected to the observation that cations are preferentially localized in the major groove of G-tract sequences. Generic DNA, which represents the vast majority of DNA sequences, has a more balanced occupation of the major and minor grooves by cations than A-tracts or G-tracts and is thereby stabilized in the canonical B-form helix. Thus, DNA secondary structure can be viewed as a tug of war between the major and minor grooves for cations, with A-tracts and G-tracts each having one groove that dominates the other for cation localization. Finally, the sequence-directed curvature caused by A-tracts and G-tracts can, in both cases, be explained by the cation-dependent mismatch of A-tract and G-tract helical structures with the canonical B-form helix of generic DNA (i.e., a cation-dependent junction model).     

Ion coordination in the amphotericin B channel.

The antifungal polyene antibiotic amphotericin B forms channels in lipid membranes that are permeable to ions, water, and nonelectrolytes. Anion, cation, and ion pair coordination in the water-filled pore of the "barrel" unit of the channels was studied by molecular dynamics simulations. Unlike the case of the gramicidin A channel, the water molecules do not create a single-file configuration in the pore, and some cross sections of the channel contain three or four water molecules. Both the anion and cation are strongly bound to ligand groups and water molecules located in the channel. The coordination number of the ions is about six. The chloride has two binding sites in the pore. The binding with water is dominant; more than four water molecules are localized in the anion coordination sphere. Three motifs of the ion coordination were monitored. The dominant motif occurs when the anion is bound to one ligand group. The ion is bound to two or three ligand groups in the less favorable configurations. The strong affinity of cations to the channel is determined by the negatively charged ligand oxygens, whose electrostatic field dominates over the field of the hydrogens. The ligand contribution to the coordination number of the sodium ion is noticeably higher than in the case of the anion. As in the case of the anion, there are three motifs of the cation coordination. The favorable one occurs when the cation is bound to two ligand oxygens. In the less favorable cases, the cation is bound to three or four oxygens. In the contact ion pair, the cation and anion are bound to two ligand oxygens and one ligand hydrogen, respectively. There exist intermediate solvent-shared states of the ion pair. The average distances between ions in these states are twice as large as that of the contact ion pair. The stability of the solvent-shared state is defined by the water molecule oriented along the electrostatic field of both ions.     

Competitive interaction of monovalent cations with DNA from 3D-RISM

The composition of the ion atmosphere surrounding nucleic acids affects their folding, condensation and binding to other molecules. It is thus of fundamental importance to gain predictive insight into the formation of the ion atmosphere and thermodynamic consequences when varying ionic conditions. An early step toward this goal is to benchmark computational models against quantitative experimental measurements. Herein, we test the ability of the three dimensional reference interaction site model (3D-RISM) to reproduce preferential interaction parameters determined from ion counting (IC) experiments for mixed alkali chlorides and dsDNA. Calculations agree well with experiment with slight deviations for salt concentrations >200 mM and capture the observed trend where the extent of cation accumulation around the DNA varies inversely with its ionic size. Ion distributions indicate that the smaller, more competitive cations accumulate to a greater extent near the phosphoryl groups, penetrating deeper into the grooves. In accord with experiment, calculated IC profiles do not vary with sequence, although the predicted ion distributions in the grooves are sequence and ion size dependent. Calculations on other nucleic acid conformations predict that the variation in linear charge density has a minor effect on the extent of cation competition.     

DNA coated with cationic fullerene derivative. A possible microwire in water.

Fullerene derivative 2 carrying pyridinium cation bound to sonicated calf thymus DNA in water. The binding ratio was 1 fullerene unit to 1 phosphate residue, giving the complex where DNA strand is seemingly coated with electron-conducting fullerenes. Cyclic voltammetry shows three-step redox couples in the complex, and the current peaks were broadened and shifted to positive side as compared to uncomplexed 2. Binding of 2 onto grooves of DNA double helix was suggested.     

Micropatterning of endothelial cells by guided stimulation with angiogenic factors

Micropatterning technology holds significant promise in the development of micro/nanomedical devices. The precise control of cell position and migration is important in several applications. For example, the optimal design of implantable devices depends on the implant material's micro-and nano-texture, which influences the response of nearby tissue, including the microvessels. Therefore, we were interested in endothelial cell positioning and colonization on specific surface domains in the size range of microvasculature. To this end, endothelial cells were seeded in microfabricated grooves and exposed to vascular endothelial growth factor (VEGF), which plays a key role in the angiogenic response. Patterned silicon wafers with grooves of 50 microm width and depth and 150 microm groove spacing were used. Each patterned region had two semicircular ports at either end, one of which was used to seed human retinal endothelial cells (HREC) and the other to house VEGF embedded in Matrigel. After 1 week, cells were fixed and analyzed by laser scanning cytometry (LSC). Our results shows that we can control HREC seeding and positioning in surface grooves and that the speed of colonization of the grooves can be manipulated by local VEGF application. We were able to quantify this effect, showing that HREC relocate inside the grooves twice as fast in response to VEGF stimulation, compared to control conditions, at a speed of 3.14 +/- 0.01 and 1.55 +/- 0.01 microm/min, respectively. Our approach could be used towards the fabrication of "designer" substrates or devices that not only allow patterned cell growth, but also permit dynamic cell repositioning.     

A laser Raman spectroscopic study of the interaction of the methylmercury cation with AMP, ADP and ATP

The interaction of the CH3Hg+ cation with adenosine 5'-monophosphate, adenosine 5'-diphosphate, and adenosine 5'-triphosphate has been studied in aqueous solution at neutral pH by laser Raman spectroscopy. Metal binding is shown to occur preferentially at the N-1 ring position of adenine, with some indication of coordination to the N-7 site and substitution of a proton on the exocyclic NH2 group of the nucleic base. Binding of the cation to phosphate groups also occurs extensively, with both the -PO2-3 and -PO-2 groups. The equilibrium constants for the binding to the phosphate groups and for N-1 coordination are approx. 70 and 600 M-1, respectively.     

Thallium-DNA complexes in aqueous solution. Major or minor groove binding

Thallium (Tl) binds to the major and minor grooves of B-DNA in the solid state (Howerton et al., Biochemistry 40, 10023-10031, 2001). The aim of this study was to examine the binding of Tl(I) cation with calf-thymus DNA in aqueous solution at physiological pH, using constant concentration of DNA (12.5 mM) and various concentrations of metal ions (0.5 to 20 mM). UV-visible and FTIR spectroscopic methods were used to determine the cation binding site, the binding constant and DNA structural variations in aqueous solution. Direct Tl bindings to guanine and thymine were evident by major spectral changes of DNA bases with overall binding constant of K = 1.40 x 10(4) M(-1) and little perturbations of the backbone phosphate group. Both major and minor groove bindings were observed with no alteration of the B-DNA conformation. At low metal concentration (0.5 mM), the number of cations bound were 10 per 1000 nucleotides, while at higher cation concentration (10 mM), this increased to 30 cations per 1000 nucleotides.     

Characterization of the ribulosebisphosphate carboxylase-carbon dioxide-divalent cation-carboxypentitol bisphosphate complex

Ribulosebisphosphate carboxylase forms a stable quaternary complex with CO2, divalent cation, and carboxypentitol bisphosphate. Incorporation of nonexchangeable CO2 into the complex requires the presence of a divalent cation. MG2+, Mn2+, or Co2+ supports stoichiometric binding of CO2 activator. When the quaternary complex is formed in the presence of saturating CO2, stoichiometric amounts of cation are bound in a nonexchangeable fashion. Incorporation of Mn2+ into an enzyme-CO2-Mn2+-carboxypentitol bisphosphate complex permitted investigation of cation environment by electron spin resonance (ESR) techniques. Measurements at 9 and 35 GHz suggest rhombic distortion of the coordination sphere of bound Mn2+. A complex inner sphere liganding of the cation bound in the quaternary complex would account for both the ESR spectra and the marked stability of the complex with respect to cation exchange.     

Data mining of metal ion environments present in protein structures

Analysis of metal-protein interaction distances, coordination numbers, B-factors (displacement parameters), and occupancies of metal-binding sites in protein structures determined by X-ray crystallography and deposited in the PDB shows many unusual values and unexpected correlations. By measuring the frequency of each amino acid in metal ion-binding sites, the positive or negative preferences of each residue for each type of cation were identified. Our approach may be used for fast identification of metal-binding structural motifs that cannot be identified on the basis of sequence similarity alone. The analysis compares data derived separately from high and medium-resolution structures from the PDB with those from very high-resolution small-molecule structures in the Cambridge Structural Database (CSD). For high-resolution protein structures, the distribution of metal-protein or metal-water interaction distances agrees quite well with data from CSD, but the distribution is unrealistically wide for medium (2.0-2.5 Å) resolution data. Our analysis of cation B-factors versus average B-factors of atoms in the cation environment reveals substantial numbers of structures contain either an incorrect metal ion assignment or an unusual coordination pattern. Correlation between data resolution and completeness of the metal coordination spheres is also found.     

A spectroscopic study of the interaction of the VO2+ cation with the two components of chondroitin sulfate

The interaction of the vanadyl (IV) cation with N-acetyl-D-galactosamine, D-galactosamine, and D-glucuronic acid has been investigated by electron absorption spectroscopy at different mental to ligand ratios and pH values. In the case of D-glucuronic acid, a more detailed study was undertaken, using differential IR spectroscopy in solution. The results show that the cation interacts with the two nitrogenated molecules only at higher pH values, generating 2∶1 lig-and to metal complexes in which coordination occurs through two pairs of deprotonated OH groups of the rings. In the case of D-glucuronic acid, the IR-measurements allowed a wider insight into the structural characteristics of the complexes generated in acidic media. The involvement of the glycosidic oxygen atom in coordination, is suggested at pH=3.     

Electron spin echo envelope modulation studies of pyruvate kinase active-site complexes

Electron spin echo envelope modulation (ESEEM) spectroscopy, with Mn2+ and VO2+ as paramagnetic probes, was used to examine active-site structures at the protein-based divalent cation site of rabbit muscle pyruvate kinase in the presence of substrates, products, and the requisite inorganic cofactors. Two different VO.protein complexes were clearly distinguished, which differed with respect to coordination of the active-site lysine to VO2+. Lysine coordination was sensitive to the presence of pyruvate and phosphoenolpyruvate (PEP) and to the nature of the monovalent cation. In the presence of MgATP and oxalate, a 4-MHz 31P contact interaction was observed, which indicates that the ATP is directly coordinated to Mn2+ at the protein-based site. No 31P contact interactions were observed, however, in the presence of PEP. Pyruvate was determined to be a bidentate ligand of VO2+, on the basis of the observation of 2.2- and 5.4-MHz 13C contact interactions between VO2+ and [1-13C]pyruvate and [2-13C]pyruvate, respectively. Magnetic coupling between VO2+ or Mn2+ and 23Na, 39K, and 133Cs was observed, demonstrating the close proximity of the monovalent cation and the protein-based divalent cation.     

Metal cation controls myosin and actomyosin kinetics

We have perturbed myosin nucleotide binding site with magnesium‐, manganese‐, or calcium‐nucleotide complexes, using metal cation as a probe to examine the pathways of myosin ATPase in the presence of actin. We have used transient time‐resolved FRET, myosin intrinsic fluorescence, fluorescence of pyrene labeled actin, combined with the steady state myosin ATPase activity measurements of previously characterized D.discoideum myosin construct A639C:K498C. We found that actin activation of myosin ATPase does not depend on metal cation, regardless of the cation‐specific kinetics of nucleotide binding and dissociation. The rate limiting step of myosin ATPase depends on the metal cation. The rate of the recovery stroke and the reverse recovery stroke is directly proportional to the ionic radius of the cation. The rate of nucleotide release from myosin and actomyosin, and ATP binding to actomyosin depends on the cation coordination number.     

Solution conformation of the C-terminal domain of skeletal troponin C. Cation, trifluoperazine and troponin I binding effects

Proton magnetic resonance spectroscopy has been used to study the cation (Mg2+, Ca2+)-dependent conformational states of the C-terminal domain of rabbit skeletal troponin C under a variety of solution conditions. Nuclear Overhauser data and paramagnetic probe observations provide definition of the configuration of this region of troponin C. Comparative study of homologous proteins identify common features of the tertiary structure relevant to the cation binding reaction. Complex formation with troponin I and the drug trifluoperazine is observed to adjust the solution conformation of the C-terminal domain of troponin C. The interactive conformational response to cation coordination and the binding of the drug and troponin I are discussed.     

EXAFS study of zinc coordination in bacitracin A.

Bacitracin is a dodecapeptide antibiotic produced by Bacillus sp. The antibacterial activity depends upon the peptide binding a divalent metal. Hitherto, the exact coordination of the cation has not been established. In particular the role played by the sulphur and nitrogen atoms of the thiazoline ring of bacitracin A has not been clear. Here the coordination of Zn2+ by bacitracin A has been studied using extended X-ray absorption fine structure. The experimental data are consistent with a model in which zinc is coordinated by one oxygen and three nitrogen atoms with the sulphur atom of the thiazoline ring not being directly involved in the zinc coordination.     

Divalent cation coordination and mode of membrane interaction in cyclotides: NMR spatial structure of ternary complex Kalata B7/Mn2+/DPC micelle

The cyclotides are the family of hydrophobic bioactive plant peptides, characterized by a circular protein backbone and three knot forming disulfide bonds. It is believed that membrane activity of the cyclotides underlines their antimicrobial, cytotoxic and hemolytic properties, but the specific interactions with divalent cations can be also involved. To assess the mode of membrane interaction and divalent cation coordination in cyclotides, the spatial structure of the Möbius cyclotide Kalata B7 from the African perennial plant Oldenlandia affinis was determined in the presence of anisotropic membrane mimetic (dodecylphosphocholine micelles). The model of peptide/cation/micelle complex was built using 5-doxylstearate and Mn²⁺ relaxation probes. Results show that the peptide binds to the micelle surface with relatively high affinity by two hydrophobic loops (loop 2 – Thr⁶-Leu⁷ and loop 5 – Trp¹⁹-Ile²¹). The partially hydrated divalent cation is coordinated by charged side-chain of Glu³, aromatic side chain of Tyr¹¹ and free carbonyls of Thr⁴ and Thr⁹, and is located in direct contact with the polar head-groups of detergent. The comparison with data about other cyclotides indicates that divalent cation coordination is the invariant property of all cyclotides, but the mode of peptide/membrane interactions is varied. Probably, the specific cation/peptide interactions play a major, but yet not known, role in the biological activity of the cyclotides.     

Pseudomonas putida cytochrome P-450. The effect of complexes of the ferric hemeprotein on the relaxation of solvent water protons.

With pulsed nuclear magnetic resonance techniques, the effects of various complexes of ferric cytochrome P-450 on the relaxation rate of bulk solution water protons have been determined. For the camphor, metyrapone, and 4-phenylimidazole complexes, the experimental results are consistent with outer sphere relaxation effects. However, for the substrate-free enzyme, the magnitude and temperature dependence of the paramagnetic relaxation effects indicate the presence of exchangeable protons in the coordination sphere of the heme iron atom. The exchange rate (9.3 x 10(4) S-1 at 25 degrees) and the thermodynamic activation parameters for the exchange process are very similar to those of acid metmyoglobin and acid methemoglobin, suggesting that a water molecule, and not an amino acid residue of the protein, coordinates to the ferric cation of the enzyme in the absence of added substrate or ligands. From the equations appropriate for coordination sphere protons, the distance between these protons and the ferric heme cation was evaluated as 2.1 A, which further supports the interpretation. These experimental results demonstrate that the solvent accessibility of the ferric cation of substrate-free cytochrome P-450 is significantly reduced by the binding of substrate or nitrogenous ligands to the hemeprotein.     

Combination of extended X-ray absorption fine structure spectroscopy with lipidic cubic phases for the study of cation binding in bacteriorhodopsin

We have performed a quantitative X-ray absorption fine structure analysis of bacteriorhodopsin in purple membrane patches and in lipidic cubic phases regenerated with Mn²⁺. Lipidic cubic phases and purple membrane results have been compared, demonstrating that the lipidic cubic phase process does not introduce relevant distortions in the local geometry of the cation binding sites. For both samples, we have observed similarities for Mn²⁺ coordination in terms of type, number, and average distances of surrounding atoms, indicating a first coordination shell composed by 6 O atoms, and 3/4 C atoms located in the second coordination shell.     

The size, shape and orientation of pore grooves in the egg shells of Rhea sp.

In the egg shells of Rhea sp. there are grooves on the surface and it is into these grooves that one or more pore mouths open.
Five egg shells of Rhea americana and four of Rhea darwinii were studied in detail in respect of the numbers of pore mouths and the number, length and orientation of the grooves. It was found that R. americana had nearly twice as many pore mouths and grooves as had R. darwinii. Their distribution in both species showed more and longer grooves and more pore mouths in the equatorial regions than in the polar regions of the shell. The grooves were also found to be approximately longitudinally orientated in the equatorial regions but this orientation became less marked towards the polar regions. Not surprisingly longer grooves tended to have more pore mouths than did shorter grooves, but, in addition, broader grooves were associated with greater length too.     

Mutagenesis of a single hydrogen bond in cytochrome P-450 alters cation binding and heme solvation

Cytochrome P-450cam, a monoxygenase responsible for the regiospecific hydroxylation of camphor, binds its substrate through complimentary van der Waals contacts and the formation of a single hydrogen bond between tyrosine 96 and the ketone group of camphor. Substrate association is positively regulated through the binding of a monovalent cation and the oxidation-reduction potential modulated by the spin state of the ferric heme controlled by water access to the sixth coordination site of the iron. Removal of this single hydrogen bond via site-directed mutagenesis of tyrosine 96 to phenylalanine 96 defines this aspect of the protein structure as responsible for the linkage between cation and substrate cooperativities, the degree of spin state conversion resulting from water access via macromolecule and substrate dynamics, and suggests a specific location for the cation binding site.