Structural studies of gellan gum,an extracellular polysaccharide elaborated by Pseudomonas elodea

The structure of gellan gum, a polysaccharide of potential commercial usefulness elaborated by Pseudomonas elodea, has been investigated. It is concluded that the polysaccharide is composed of tetrasaccharide repeating-units having the following structure.

Of the repeating units, ～25% contain an O-acetyl group linked to C-6 of one of the β-d-glucopyranosyl residues.     

Binding of Cu(2+)-beta-cyclodextrin complex to calf thymus DNA.

Cu(2+)-beta-cyclodextrin (1:1) complex has been found by UV, fluorescence and CD spectroscopy, polarimetry and gel electrophoresis to bind reversibly to calf thymus DNA. Using UV the binding constant was found to be 45280 +/- 7100 M-1. The binding of the complex Cu(2+)-BCD with DNA was stronger than that of free Cu2+. However the ternary complex formed thus was destroyed by EDTA.     

Na~(+)、Ca~(2+)、Cu~(2+)和Zn~(2+)与HSA相互作用的~(23)(Na)-NMR研究

 本文应用~23Na-NMR波谱技术,研究了Na~(+)、Ca~(2+)、Cu~(2+)和Zn~(2+)与人体血清白蛋白(HSA)的相互作用。在实验基础上,通过引入两位快交换模型,拟合计算获得了Na~(+)与HSA相互作用的结合常数和处于结合状态Na~(+)的相关时间;实验表明Ca~(2+)能与Na~(+)竞争同HSA结合,拟合计算获得了两者与HSA相互作用结合常数的比值,棕榈酸钠能增强Ca~(2+)同Na~(+)竞争与HSA结合的能力;从实验上未能观察到Cu~(2+)、Zn~(2+)能同Na~(+)竞争与HSA相互作用的证据。     

Pyrrolidine dithiocarbamate-induced apoptosis depends on cell type, density, and the presence of Cu(2+) and Zn(2+)

Pyrrolidine dithiocarbamate (PDTC) has been found toinduce or inhibit apoptosis in different cell types. Here we show that PDTC dose-dependently reduced the viability of rat smooth muscle cells(rSMC), human fibroblasts, and endothelial cells at low but not at highcell density. Endothelial cells were least sensitive, fibroblastsshowed a medium sensitivity, and rSMC showed a high sensitivity toPDTC-mediated cell death. An early reduction in the mitochondrialmembrane potential indicated a rapid onset of apoptosis in rSMC.Apoptosis was further confirmed by annexin V staining and DNAfragmentation analysis. Gel shift analysis demonstrated increasednuclear factor (NF)-B activity in high-density rSMC compared withlow-density cells. NF-B has recently been shown to regulate theinduction of anti-apoptotic proteins. Although PDTC is widely used asan inhibitor for NF-B and a radical scavenger, our data show thatPDTC rather enhanced NF-B activity and, alone or in combination withmenadione, induced oxygen radical generation. Notably, PDTC failed toreduce rSMC viability in medium without Cu²⁺ orZn²⁺, and addition of Cu²⁺ or Zn²⁺resulted in a dose-dependent increase in PDTC-induced cell death. Addition of both Cu²⁺ and Zn²⁺ showedsynergistic effects. Our results indicate that the induction ofapoptosis by PDTC requires Cu²⁺ and Zn²⁺ and isdependent on cell type and density. Such differential effects may haveimplications for studies of PDTC as an anti-atherosclerotic orimmunomodulatory drug.

     

DsRed as a highly sensitive, selective, and reversible fluorescence-based biosensor for both Cu(+) and Cu(2+) ions

The wild type form of Red fluorescent protein (DsRed), an intrinsically fluorescent protein found in tropical corals, is found to be highly selective, reversible and sensitive for both Cu(+) and Cu(2+), with a nanomolar detection limit. The selectivity towards these ions is retained even in the presence of other heavy metal ions. The K(d) values for monovalent and divalent copper, based on single binding isotherms, are 450 and 540 nM, respectively. The wild type DsRed sensitivity to Cu(2+) (below 1 ppb) is seven orders of magnitude better than that of the related wild type Green Fluorescent protein (GFP), and it is even 40 times more sensitive than engineered mutants of GFP. Potential binding sites have been proposed, based on amino acid sequences for copper binding and the distance from the chromophore, with the aid of computer modeling.     

Urea effect on Cu(2+)-induced DNA structural transitions in solution

Cu(2+) ion interaction with DNA in aqueous solutions containing urea (0-5 M) was studied by IR spectroscopy. It was shown that upon the Cu(2+) ion binding DNA transition into a compact form occurs. This transition is of positive cooperativity. We suppose that the mechanism of Cu(2+)-induced DNA compaction in solutions containing urea is not completely electrostatic. Urea addition to the DNA solution decreases the Cu(2+) ion concentration required to induce DNA compaction. As the urea content in solution rises, the binding constant of Cu(2+) ions interacting with DNA increases, going through the maximum in the case of 2 M solution; further increase of the urea content in solutions leads to decrease of the binding constant. DNA transition into the compact form under the Cu(2+) ion action is determined not only by the effects of the solution dielectric permeability but by the solvation effects; when changes of the dielectric permeability are small the solvation effects may prevail. Urea addition to the DNA solution also decreases cooperativity of the DNA compaction process. Perhaps, cooperativity of the DNA transition into the compact state depends on the ordered spatial structure of water adjacent to the macromolecule and decreases on the structure destruction.     

Mg2+ as a trigger of condenstion-decondensation transition of chromatin during mitosis

It is proposed that the direct trigger of the mitotic condensation-decondensation transition of interphase chromatin is the elevation and subsequent normalization of cellular concentration of ionized magnesium. The hypothesis is based on the analysis of structural changes of chromatin during condensation and their relation to known experimental data on mitotic condensation and mitosis.     

Synthesis and characterization of N-(2-aminoethyl)-2-acetamidyl gellan gum with potential biomedical applications

N-(2-aminoethyl)-2-acetamidyl gellan gum (GCM-EDA) was prepared by carboxymethylation (via nucleophilic substitution of primary hydroxyl groups of the β-d-glucose unit of gellan gum, in the presence of alkali and chloroacetic acid) and reaction with tert-butyl N-(2-aminoethyl) carbamate (N-Boc-EDA) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) as an activator, followed by deprotection with trifluoroacetic acid. The structural confirmation and characterization of N-(2-aminoethyl)-2-acetamidyl gellan gum was performed by spectroscopic, rheological and thermogravimetric analysis, and in vitro tests showed a lack of cytotoxicity which is indicative of the potential of this material to be used in biomedical applications.     

Unusually High-Affinity Mg(2+) Binding at the AU-Rich Sequence within the Antiterminator Hairpin of a Mg(2+) Riboswitch

Mg(2+) -Responsive riboswitches represent a fascinating example of bifunctional RNAs that sense Mg(2+) ions with high selectivity and autonomously regulate the expression of Mg(2+) -transporter proteins. The mechanism of the mgtA riboswitch is scarcely understood, and a detailed structural analysis is called for to study how this RNA can selectively recognize Mg(2+) and respond by switching between two alternative stem loop structures. In this work, we investigated the structure and Mg(2+) -binding properties of the lower part of the antiterminator loop C from the mgtA riboswitch of Yersinia enterocolitica by solution NMR and report a discrete Mg(2+) -binding site embedded in the AU-rich sequence. At the position of Mg(2+) binding, the helical axis exhibits a distinct kink accompanied by a widening of the major groove, which accommodates the Mg(2+) -binding pocket. An unusually large overlap between two adenine residues on the opposite strands suggests that the bending may be sequence-induced by strong stacking interactions, enabling Mg(2+) to bind at this so-far not described metal-ion binding site.     

Electron spin-echo envelope modulation study of multicrystalline Cu(2+)-insulin: effects of Cd(2+) on the nuclear quadrupole interaction of the Cu(2+)-coordinated imidazole remote nitrogen

A comparison of electron spin-echo envelope modulation (ESEEM) spectra from multi-crystalline Cu(2+)-insulin with and without additional Cd(2+) show a dramatic change in the quadrupole coupling parameters of the remote nitrogens of the two histidine imidazoles that ligate to copper. Without Cd(2+), the quadrupole parameters are like those observed in blue copper proteins and in copper substituted lactoferrin. With Cd(2+) soaked into the Cu(2+)-insulin crystals, the quadrupole parameters are similar to those found in galactose oxidase. Theoretical simulations of ESEEM spectra guided by structure modeling suggest that these changes originate from differences in the hydrogen bonding environments of the imidazole remote nitrogen. In addition, a compilation of results from previous ESEEM studies of copper proteins reveals that the asymmetry parameter, eta, may be an indicator of type of hydrogen bond the imidazole remote nitrogen makes. When eta > or = 0.9, the nitrogen hydrogen bonds to water, whereas when eta < 0.9, the nitrogen hydrogen bonds to the protein.     

Interaction of Cu(2+) with His-Val-His and of Zn(2+) with His-Val-Gly-Asp, two peptides surrounding metal ions in Cu,Zn-superoxide dismutase enzyme

His-Val-His and His-Val-Gly-Asp are two naturally occurring peptide sequences, present at the active site of Cu,Zn-superoxide dismutase (Cu,Zn-SOD). The interactions of His-Val-His=A (copper binding site) with Cu(II) and of His-Val-Gly-Asp=B (zinc binding site) with Zn(II) have been studied by using both potentiometric and spectroscopic methods (visible, EPR, NMR). The stoichiometry, stability constants and solution structure of the complexes formed have been determined. The binding modes of the species [CuAH](2+) and [CuA](+) were characterized by histamine type of coordination. [CuA](+) is further stabilized by the formation of a macrochelate with the involvement of the imidazole of the C-terminal histidine. The existence of macrochelate results in a slight distortion of the coordination geometry providing good base for the development of enzyme models. The enhanced stability of the macrochelate suppresses the formation of bis-complexes as well as the amide deprotonation. This process, however, takes place at higher pH resulting in the formation of the 4 N(-) coordinated [NH(2),N(-),N(-),N(im)] species [CuAH(2-)](-). On the other hand, in the case of the Zn(II)-His-Val-Gly-Asp system, coordination takes place at the terminal carboxylate in species [ZnBH(2)](2+). Monodentate binding occurs via the N-terminal imidazole in [ZnBH](+) while histamine type of coordination is possible in [ZnB], [ZnB(2)H](-) and [ZnB(2)](2-) species. Amide deprotonation does not take place in the case of Zn(2+), hydroxo-complexes are formed instead.     

Comparative analysis of the behavior of gellan gum (S-60) and welan gum (S-130) in dilute aqueous solution

Optical rotation, circular dichroism, and microcalorimetric data clearly and consistently show that gellan gum, S-60 (Me₄N⁺ form), undergoes in water at 25° a rather sharp conformational transition upon increasing the concentration of added Me₄NCl. Similar data show that S-60 behaves anomalously upon addition of Ca²⁺ ions with, eventually, formation of aggregates and/or gels. The Me₄NCl-induced conformational change of S-60 is thermally reversible with no hysteresis. In contrast, with welan gum, S-130 (Me₄N⁺ form), no evidence could be found for a dependence of chain conformation of the main external variables considered. Comparison of the circular-dichroism spectra of the two polysaccharides suggests that S-130 in water might be present in a stiff conformation similar to that assumed by S-60 in aqueous Me₄NCl.     

Structural features of the Cu(2+)-vancomycin complex

The structure of vancomycin coordinated to Cu(2+) ions is presented and structural aspects upon metal coordination are discussed. The asymmetric part of unit cell comprises two independent molecules of vancomycin-Cu(2+) complex, one of them is partially disordered. The binding site involves one imino nitrogen atom, two amide nitrogen atoms delivered by peptide bonds, and carboxyl oxygen from the peptide moiety. The identical set of donor atoms is not reflected in identical coordination geometry around individual metal ions. The studied complex presents two distinct types of conformation. Additionally, leucinyl side chain in one conformer is disordered leading to another type of conformation. The complex molecules form heterodimer with antiparallel hydrogen bonding.     

Structural exploration of viral matrix protein 40 interaction with the transition metal ions (Ag+ and Cu2+)

Here, a theoretical and comprehensive study of the structural features and interaction properties of viral protein 40 is being briefed out to understand the mechanism of Ebola virus (EV) with structural and orbital analysis. In general, viral protein 40 is the key protein for the oligomerization, the N-terminal loop region in the viral protein 40 and it is essential for the viral replication in Ebola. The electronic structures of native N-terminal loop (His124-Asn134) and metalized (Mⁿ⁺=Ag⁺ and Cu²⁺) complexes are optimized at the M06-2X/LANL2DZ level of theory. Among Mⁿ⁺-interacted N-loop complexes, Cu²⁺-interacted N-terminal loop complex has the highest interaction energy of –973.519?kcal/mol and also it has the stabilization energy in the range of 9.92?kcal/mol. The cation-π interactions between His124, Pro131 and Arg134 residues are the important factor, which enhances the interaction energy of viral protein 40. Due to the chelation behavior of metal ions, the backbone and the side chains of N-terminal loop regions are deviated from the planarity that results in the formation of classical hydrogen bonds between N-terminal loop regions. Molecular dynamics simulation studies also revealed that the structural transformations of Nloop into a stable α-helix and β-sheet folded conformations due to the interaction of Ag⁺ and Cu²⁺ ions in the N-terminal loop region. The hydrogen bond formation and hydrophobic interactions are responsible for the stability and structural changes in N-terminal loop region. Therefore, it is clear that interaction of metal ion with viral protein-40 reduces the replication of the disease by inducing the secondary structural changes.

Communicated by Ramaswamy H. Sarma     

PLC zeta: a sperm-specific trigger of Ca(2+) oscillations in eggs and embryo development

Upon fertilisation by sperm, mammalian eggs are activated by a series of intracellular Ca(2+) oscillations that are essential for embryo development. The mechanism by which sperm induces this complex signalling phenomenon is unknown. One proposal is that the sperm introduces an exclusive cytosolic factor into the egg that elicits serial Ca(2+) release. The 'sperm factor' hypothesis has not been ratified because a sperm-specific protein that generates repetitive Ca(2+) transients and egg activation has not been found. We identify a novel, sperm-specific phospholipase C, PLC zeta, that triggers Ca(2+) oscillations in mouse eggs indistinguishable from those at fertilisation. PLC zeta removal from sperm extracts abolishes Ca(2+) release in eggs. Moreover, the PLC zeta content of a single sperm was sufficient to produce Ca(2+) oscillations as well as normal embryo development to blastocyst. Our results are consistent with sperm PLC zeta as the molecular trigger for development of a fertilised egg into an embryo.     

Binding of cadmium (Cd2+) to E-CAD1, a calcium-binding polypeptide analog of E-cadherin

Recent studies have shown that Cd²⁺ can damage the Ca²⁺-dependent junctions between renal epithelial cells in culture, and preliminary evidence suggests that this effect may involve the interaction of Cd²⁺ with E-cadherin, a Ca²⁺-dependent cell adhesion molecule that is localized at the adhering junctions of epithelial cells. To determine whether or not Cd²⁺ might bind directly to the E-cadherin molecule, we studied the binding of Cd²⁺ to E-CAD1, a recombinant, 145-residue polypeptide that corresponds to one of the extracellular Ca²⁺-binding regions of mouse E-cadherin. By using an equilibrium microdialysis technique, we were able to show that Cd²⁺ could, in fact, bind to E-CAD1. The binding was saturable, with a maximum of one Cd²⁺ binding site per E-CAD1 molecule. The apparent dissociation constant (K_D) for the binding was about 20 μM, a concentration similar to that which has been shown to disrupt the junctions between epithelial cells. Other results showed that the binding of Cd²⁺ was greatly reduced when excess Ca²⁺ was included in the dialysis solution. These results suggest that Cd²⁺ can interact with the Ca²⁺ binding regions on the E-CAD1 molecule, and they provide additional support for the hypothesis that E-cadherin might be a molecular target for Cd²⁺ toxicity.     

Role of the Na(+)-Ca(2+) exchanger as an alternative trigger of CICR in mammalian cardiac myocytes

Ca(2+) influx through the L-type Ca(2+) channels is the primary pathway for triggering the Ca(2+) release from the sarcoplasmic reticulum (SR). However, several observations have shown that Ca(2+) influx via the reverse mode of the Na(+)-Ca(2+) exchanger current (I(Na-Ca)) could also trigger the Ca(2+) release. The aim of the present study was to quantitate the role of this alternative pathway of Ca(2+) influx using a mathematical model. In our model 20% of the fast sodium channels and the Na(+)-Ca(2+) exchanger molecules are located in the restricted subspace between the sarcolemma and the SR where triggering of the calcium-induced calcium release (CICR) takes place. After determining the strengths of the alternative triggers with simulated voltage-clamps in varied membrane voltages and resting [Na](i) values, we studied the CICR in simulated action potentials, where fast sodium channel current contributes [Na](i) of the subspace. In low initial [Na](i) the Ca(2+) influx via the L-type Ca(2+) channels is the major trigger for Ca(2+) release from the SR, and the Ca(2+) influx via the reverse mode of the Na(+)-Ca(2+) exchanger cannot trigger the CICR. However, depending on the initial [Na](i), the contribution of the Ca(2+) entry via the exchanger may account for 25% (at [Na](i) = 10 mM) to nearly 100% ([Na](i) = 30 mM) of the trigger Ca(2+). The shift of the main trigger from L-type calcium channels to the exchanger reduced the delay between the action potential upstroke and the intracellular calcium transient. This may contribute to the function of the myocyte in physiological situations where [Na](i) is elevated. These main results remain the same when using different estimates for the most crucial parameters in the modeling or different models for the exchanger.     

Direct computation of long time processes in peptides and proteins: reaction path study of the coil-to-helix transition in polyalanine.

The MaxFlux reaction path algorithm was used to isolate optimal transition pathways for the coil-to-helix transition in polyalanine. Eighteen transition pathways, each connecting one random coil configuration with an ideal alpha-helical configuration, were computed and analyzed. The transition pathway energetics and mechanism were analyzed in terms of the progression of the peptide nonbonded contact formation, helicity, end-to-end distance and energetics. It was found that (1) localized turns characterized by i, i + 3 hydrogen bonds form in the early stages of the coil-to-helix transition, (2) the peptide first collapses and then becomes somewhat more extended in the final stage of helix formation, and (3) 310-helix formation does not appear to be a necessary step in the transition from coil to helix. These conclusions are in agreement with the results of more computationally intensive direct molecular dynamics simulations. Proteins 1999;36:249-261.     

The permeability transition pore as a Ca(2+) release channel: new answers to an old question

Mitochondria possess a sophisticated array of Ca(2+) transport systems reflecting their key role in physiological Ca(2+) homeostasis. With the exception of most yeast strains, energized organelles are endowed with a very fast and efficient mechanism for Ca(2+) uptake, the ruthenium red (RR)-sensitive mitochondrial Ca(2+) uniporter (MCU); and one main mechanism for Ca(2+) release, the RR-insensitive 3Na(+)-Ca(2+) antiporter. An additional mechanism for Ca(2+) release is provided by a Na(+) and RR-insensitive release mechanism, the putative 3H(+)-Ca(2+) antiporter. A potential kinetic imbalance is present, however, because the V(max) of the MCU is of the order of 1400nmol Ca(2+)mg(-1) proteinmin(-1) while the combined V(max) of the efflux pathways is about 20nmol Ca(2+)mg(-1) proteinmin(-1). This arrangement exposes mitochondria to the hazards of Ca(2+) overload when the rate of Ca(2+) uptake exceeds that of the combined efflux pathways, e.g. for sharp increases of cytosolic [Ca(2+)]. In this short review we discuss the hypothesis that transient opening of the Ca(2+)-dependent permeability transition pore may provide mitocondria with a fast Ca(2+) release channel preventing Ca(2+) overload. We also address the relevance of a mitochondrial Ca(2+) release channel recently discovered in Drosophila melanogaster, which possesses intermediate features between the permeability transition pore of yeast and mammals.