How can Ca2+ selectively activate recoverin in the presence of Mg2+? Surface plasmon resonance and FT-IR spectroscopic studies

We investigated the relationship between metal ion selective conformational changes of recoverin and its metal-bound coordination structures. Recoverin is a 23 kDa heterogeneously myristoylated Ca(2+)-binding protein that inhibits rhodopsin kinase. Upon accommodating two Ca(2+) ions, recoverin extrudes a myristoyl group and associates with the lipid bilayer membrane, which was monitored by the surface plasmon resonance (SPR) technique. Large changes in SPR signals were observed for Sr(2+), Ba(2+), Cd(2+), and Mn(2+) as well as Ca(2+), indicating that upon binding to these ions, recoverin underwent a large conformational change to extrude the myristoyl group, and thereby interacted with lipid membranes. In contrast, no SPR signal was induced by Mg(2+), confirming that even though it accommodates two Mg(2+) ions, recoverin does not induce the large conformational change. To investigate the coordination structures of metal-bound Ca(2+) binding sites, FT-IR studies were performed. The EF-hands, Ca(2+)-binding regions each comprising 12 residues, arrange to coordinate Ca(2+) with seven oxygen ligands, two of which are provided by a conserved bidentate Glu at the 12th relative position in the EF-hand. FT-IR analysis confirmed that Sr(2+), Ba(2+), Cd(2+), and Mn(2+) were coordinated to COO(-) of Glu by a bidentate state as well as Ca(2+), while coordination of COO(-) with Mg(2+) was a pseudobridging state with six-coordinate geometry. These SPR and FT-IR results taken together reveal that metal ions with seven-coordinate geometry in the EF-hands induce a large conformational change in recoverin so that it extrudes the myristoyl group, while metal ions with six-coordinate geometry in the EF-hands such as Mg(2+) remain the myristoyl group sequestered in recoverin.     

Nuclear magnetic resonance in environmental engineering: Principles and applications

This paper gives an introduction to nuclear magnetic resonance spectroscopy (NMR) and magnetic resonance imaging (MRI) in relation to applications in the field of environmental science and engineering. The underlying principles of high resolution solution and solid state NMR, relaxation time measurements and imaging are presented. Then, the use of NMR is illustrated and reviewed in studies of biodegradation and biotransformation of soluble and solid organic matter, removal of nutrients and xenobiotics, fate of heavy metal ions, and transport processes in bioreactor systems.     

Metal binding and structure-activity relationship of the metalloantibiotic peptide bacitracin

Bacitracin is a widely used metallopeptide antibiotic produced by Bacillus subtilis and Bacillus licheniformis with a potent bactericidal activity directed primarily against Gram-positive organisms. This antibiotic requires a divalent metal ion such as Zn(2+) for its biological activity, and has been reported to bind several other transition metal ions, including Mn(2+), Co(2+), Ni(2+), and Cu(2+). Despite the widespread use of bacitracin since its discovery in the early 1940s, the structure-activity relationship of this drug has not been established and the coordination chemistry of its metal complexes was not fully determined until recently. This antibiotic has been suggested to influence cell functioning through more than one route. Since bacterial resistance against bacitracin is still rare despite several decades of widespread use, this antibiotic can serve as an ideal lead for the design of potent peptidyl antibiotics lacking bacterial resistance. In this review, the results of physical (including NMR, EPR, and EXAFS) and molecular biological studies regarding the synthesis and structure of bacitracin, the coordination chemistry of its metal derivatives, the mechanism of its antibiotic actions, its influence on membrane function, and its structure and function relationship are discussed.     

Liquid crystalline phase behavior of high molecular weight DNA: a comparative study of the influence of metal ions of different size, charge and binding mode

The ability of Li(+), Na(+), K(+), Rb(+), Cs(+), Mg(2+), Ca(2+), Sr(2+), Ba(2+), Cu(2+), Cd(2+), Al(3+), V(4+), Hg(2+), Pd(2+), Au(3+), and Pt(4+) to provoke liquid crystalline (LC) phases in high molecular weight DNA was investigated. The alkali and alkaline earth metal ions provoked typical cholesteric/columnar structures, whereas transition metal ions precipitated DNA into solid/translucent gel-like aggregates. Heavy metal ions reduced viscosity of DNA solution, disrupting rigid, rod-like DNA structure necessary for LC textures. Three-layer quantum mechanical-molecular mechanical (QM/MM) studies of Li(+), Na(+), K(+), Mg(2+), and Ca(2+) binding DNA fragment suggested several possible binding modes of these ions to the phosphate groups. The dianion mode of metal binding, involving the phosphate groups of both strands of DNA, allowed for higher DNA binding affinity of the alkaline earth metal ions. These results have implications in understanding the biological role of metal ions and developing DNA-based sensors and nanoelectronic devices.     

In silico and in vitro studies of transition metal complexes derived from curcumin–isoniazid Schiff base

Abstract

A series of transition metal complexes have been synthesized from biologically active curcumin and isoniazid Schiff base. They are characterized by various spectral techniques like UV–Vis, Fourier transform infrared (FT-IR), nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR) and mass spectroscopies. Moreover, elemental analysis, magnetic susceptibility and molar conductivity measurements are also carried out. All these data evidence that the metal complexes acquire square planar except zinc(II) which adopts a tetrahedral geometry, and they are non-electrolytic in nature. Groove mode of binding between the calf thymus DNA (CT DNA) and metal complexes is confirmed by electronic absorption titration, viscosity and cyclic voltammetry studies. In addition to that, all the metal complexes are able to cleave pUC 19 DNA. Optimized geometry and ground-state electronic structure calculations of all the synthesized compounds are established out by density functional theory (DFT) using B3LYP method which theoretically reveals that copper(II) complex explores higher stability and higher biological accessibility. This is experimentally corroborated by antimicrobial studies. In silico Absorption, Distribution, Metabolism, Excretion (ADME) studies reveal the biological potential of all synthesized complexes, and also biological activity of the ligand is predicted by PASS online biological activity prediction software. Molecular docking studies are also carried out to confirm the groove mode of binding and receptor–complex interactions.     

Metal ion selectivity for formation of the calmodulin-metal-target peptide ternary complex studied by surface plasmon resonance spectroscopy.

Ion selectivities for Ca(2+) signaling pathways of 33 metal ions were examined based on the Ca(2+)-dependent on/off switching mechanism of calmodulin (CaM): Ca(2+) ion-induced selective binding of CaM-Ca(2+) ion complex to the target peptide was observed as an increase in surface plasmon resonance (SPR) signals. As the target peptide, M13 of 26-amino-acid residues derived from skeletal muscle myosin light-chain kinase was immobilized in the dextran matrix, over which sample solutions containing CaM and each metal ion were injected in a flow system. Large changes in SPR signals were also observed for Sr(2+), Ba(2+), Cd(2+), Pb(2+), Y(3+) and trivalent lanthanide ions, thereby indicating that not only Ca(2+) but also these metal ions induce the formation of CaM-M13-metal ion ternary complex. No SPR signal was, however, induced by Mg(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+) and all monovalent metal ions examined. The latter silent SPR signal indicates that these ions, even if they bind to CaM, are incapable of forming the CaM-M13-metal ion ternary complex. Comparing the obtained SPR results with ionic radii of those metal ions, it was found that all cations examined with ionic radii close to or greater than that of Ca(2+) induced the formation of the CaM-metal-M13 ternary complex, whereas those with smaller ionic radii were not effective, or much less so. Since these results are so consistent with earlier systematic data for the effects of various metal ions on the conformational changes of CaM, it is concluded that the present SPR analysis may be used for a simple screening and evaluating method for physiologically relevant metal ion selectivity for the Ca(2+) signaling via CaM based on CaM/peptide interactions.     

Colorimetric and fluorimetric assays to quantitate micromolar concentrations of transition metals

Transition metal ions, although maintained at low concentrations, play diverse important roles in many biological processes. Two assays useful for the rapid quantification of a range of first-row transition metal ions have been developed. The colorimetric assay extends the 4-(2-pyridylazo)resorcinol assay of Hunt et al. (J. Biol. Chem. 255, 14793 (1984)) to measure nanomole quantities of Co(2+), Ni(2+), and Cu(2+) as well as Zn(2+). The fluorimetric assay takes advantage of the coordination of a number of metal ions (Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+)) by Fura-2 and can also be used to measure nanomole quantities of these ions. The assays developed here have the advantage of not requiring the extensive sample preparation necessary for other methodologies, such as atomic absorption spectroscopy and inductively coupled plasma emission spectroscopy (ICPES), while being comparable in accuracy to the detection limits of ICPES for the first-row transition metal ions. To demonstrate the effectiveness of these assays, we determined the affinity of carbonic anhydrase II (CA II), a prototypical zinc enzyme, for Ni(2+) and Cd(2+). These data indicate that CA II binds transition metals with high affinity and is much more selective for Zn(2+) over Ni(2+) or Cd(2+) than most small-molecule chelators or other metalloenzymes.     

The metallomics approach: use of Fe(II) and Cu(II) footprinting to examine metal binding sites on serum albumins

Metal binding to serum albumins is examined by oxidative protein-cleavage chemistry, and relative affinities of multiple metal ions to particular sites on these proteins were identified using a fast and reliable chemical footprinting approach. Fe(ii) and Cu(ii), for example, mediate protein cleavage at their respective binding sites on serum albumins, in the presence of hydrogen peroxide and ascorbate. This metal-mediated protein-cleavge reaction is used to evaluate the binding of metal ions, Na(+), Mg(2+), Ca(2+), Al(3+), Cr(3+), Mn(2+), Co(2+), Ni(2+), Zn(2+), Cd(2+), Hg(2+), Pb(2+), and Ce(3+) to albumins, and the relative affinities (selectivities) of the metal ions are rapidly evaluated by examining the extent of inhibition of protein cleavage. Four distinct systems Fe(II)/BSA, Cu(II)/BSA, Fe(II)/HSA and Cu(II)/HSA are examined using the above strategy. This metallomics approach is novel, even though the cleavage of serum albumins by Fe(II)/Cu(II) has been reported previously by this laboratory and many others. The protein cleavage products were analyzed by SDS PAGE, and the intensities of the product bands quantified to evaluate the extent of inhibition of the cleavage and thereby evaluate the relative binding affinities of specific metal ions to particular sites on albumins. The data show that Co(II) and Cr(III) showed the highest degree of inhibition, across the table, followed by Mn(II) and Ce(III). Alakali metal ions and alkaline earth metal ions showed very poor affinity for these metal sites on albumins. Thus, metal binding profiles for particular sites on proteins can be obtained quickly and accurately, using the metallomics approach.     

Insights into metalloproteins and metallodrugs from electron paramagnetic resonance spectroscopy

Metal ions play an important role in diverse biological processes, and much of the basic knowledge derived from studying native bioinorganic systems are applied in the synthesis of new molecules with the aim of diagnosing and treating diseases. At first glance, metalloproteins and metallodrugs are very different systems, but metal ion coordination, redox chemistry and substrate binding play essential roles in advancing both of these research fields. In this article, we discuss recent metalloprotein and metallodrug studies where electron paramagnetic resonance spectroscopy served as a major tool to gain a better understanding of metal-based structures and their function.     

Multidimensional NMR methods for protein structure determination

Structural studies of proteins are critical for understanding biological processes at the molecular level. Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for obtaining structural and dynamic information on proteins and protein-ligand complexes. In the present review, methodologies for NMR structure determination of proteins and macromolecular complexes are described. In addition, a number of recent advances that reduce the molecular weight limitations previously imposed on NMR studies of biomolecules are discussed, highlighting applications of these technologies to protein systems studied in our laboratories.     

Development of magnetically aligned phospholipid bilayers in mixtures of palmitoylstearoylphosphatidylcholine and dihexanoylphosphatidylcholine by solid-state NMR spectroscopy

This study reports the solid-state NMR spectroscopic characterization of a long chain phospholipid bilayer system which spontaneously aligns in a static magnetic field. Magnetically aligned phospholipid bilayers or bicelles are model systems which mimic biological membranes for magnetic resonance studies. The oriented membrane system is composed of a mixture of the bilayer forming phospholipid palmitoylstearoylphosphatidylcholine (PSPC) and the short chain phospholipid dihexanoylphosphatidylcholine (DHPC) that breaks up the extended bilayers into bilayered micelles or bicelles that are highly hydrated (approx. 75% aqueous). Traditionally, the shorter 14 carbon chain phospholipid dimyristoylphosphatidylcholine (DMPC) has been utilized as the bilayer forming phospholipid in bicelle studies. Alignment (perpendicular) was observed with a PSPC/DHPC q ratio between 1.6 and 2.0 slightly above T(m) at 50 degrees C with (2)H and (31)P NMR spectroscopy. Paramagnetic lanthanide ions (Yb(3+)) were added to flip the bilayer discs such that the bilayer normal was parallel with the static magnetic field. The approx. 1.8 (PSPC/DHPC) molar ratio yields a thicker membrane due to the differences in the chain lengths of the DMPC and PSPC phospholipids. The phosphate-to-phosphate thickness of magnetically aligned PSPC/DHPC phospholipid bilayers in the L(alpha) phase may enhance the activity and/or incorporation of different types of integral membrane proteins for solid-state NMR spectroscopic studies.     

Chelation ability of spironaphthoxazine with metal ions in silica gel

Spironaphthoxazine (SNO) and three metal ions, Mg(2+), Zn(2+), and Al(3+), were dispersed in silica gels by the sol-gel method. The chelation ability of SNO with the metal ions in silica gels was investigated by measuring the fluorescence spectra and was compared to that of 8-hydroxyquinoline (8-HQ) in ethanol and silica gels. A merocyanine-type isomer photoderived from SNO as well as 8-HQ easily formed complexes of the metal ions in the order of Al(3+), Zn(2+), and Mg(2+) because the coordination ability of the metal ions to such ligands depended on their electron affinity. The changes in the fluorescence spectra of the silica gel samples during light irradiation were also investigated. The relative band intensity due to the intermediate species between the original SNO and the merocyanine species decreased and that of the complex increased with the UV irradiation time. The reverse process was observed during visible irradiation. The UV irradiation effects on the chelation of SNO and its photochromic property also depended on the electron affinity of the metal ions.     

Sensitive label-free oligonucleotide-based microfluidic detection of mercury (II) ion by using exonuclease I

Mercury is a highly toxic metal that can cause significant harm to humans and aquatic ecosystems. This paper describes a novel approach for mercury (Hg(2+)) ion detection by using label-free oligonucleotide probes and Escherichia coli exonuclease I (Exo I) in a microfluidic electrophoretic separated platform. Two single-stranded DNAs (ssDNA) TT-21 and TT-44 with 7 Thymine-Thymine mispairs are employed to capture mercury ions. Due to the coordination structure of T-Hg(2+)-T, these ssDNAs are folded into hairpin-like double-stranded DNAs (dsDNA) which are more difficult to be digested by Exo I, as confirmed by polyacrylamide gel electrophoresis (PAGE) analysis. A series of microfluidic capillary electrophoretic separation studies are carried out to investigate the effect of Exo I and mercury ion concentrations on the detected fluorescence intensity. This method has demonstrated a high sensitivity of mercury ion detection with the limit of detection around 15 nM or 3 ppb. An excellent selectivity of the probe for mercury ions over five interference ions Fe(3+), Cd(2+), Pb(2+), Cu(2+) and Ca(2+) is also revealed. This method could potentially be used for mercury ion detection with high sensitivity and reliability.     

Metal-phosphate interactions in the hammerhead ribozyme observed by 31P NMR and phosphorothioate substitutions

The hammerhead ribozyme is a catalytic RNA that requires divalent metal cations for activity under moderate ionic strength. Two important sites that are proposed to bind metal ions in the hammerhead ribozyme are the A9/G10.1 site, located at the junction between stem II and the conserved core, and the scissile phosphate (P1.1). (31)P NMR spectroscopy in conjunction with phosphorothioate substitutions is used in this study to investigate these putative metal sites. The (31)P NMR feature of a phosphorothioate appears in a unique spectral window and can be monitored for changes upon addition of metals. Addition of 1-2 equiv of Cd(2+) to the hammerhead with an A9-S(Rp) or A9-S(S)(Rp) substitution results in a 2-3 ppm upfield shift of the (31)P NMR resonance. In contrast, the P1.1-S(Rp) and P1.1-S(Sp) (31)P NMR features shift slightly and in opposite directions, with a total change in delta of 相似文献   

Fluorescence study of the interaction of Suwannee River fulvic acid with metal ions and Al3+-metal ion competition

In this study emission and synchronous-scan fluorescence spectroscopy have been used to investigate the interaction of the class A (oxygen seeking 'hard acid') metal Al(3+), with Suwannee River fulvic acid (SRFA), as well as competition between Al(3+) and several other metal ions (Ca(2+), Mg(2+), Cu(2+), Pd(2+), La(3+), Tb(3+) and Fe(3+)) for binding sites on SRFA. Of the four metal ions possessing very similar (and relatively low) ionic indices (Ca(2+), Mg(2+), Cu(2+) and Pd(2+)) only the latter two paramagnetic ions significantly quenched SRFA fluorescence emission intensity. Of the four metal ions possessing very similar (and relatively low) covalent indices (Ca(2+), Mg(2+), La(3+) and Tb(3+)) only the last paramagnetic ion (Tb(3+)) significantly quenched SRFA fluorescence. None of these metals was able to significantly compete with SRFA-bound Al(3+).Fe(3+), which differs substantially from all of the other metals examined in this study in that it possesses a relatively high ionic index (but not as high as Al(3+)) and a relatively low covalent index (but not as low as Al(3+)), was able not only to quench SRFA fluorescence but also to compete (at least to some extent) with SRFA-bound Al(3+). Synchronous-scan fluorescence SRFA spectra taken in the absence and presence of Fe(3+) and/or Al(3+) support the view that these two metal ions can compete for sites on SRFA. The results of these fluorescence experiments further confirm the Al(3+), and metal ions that have electronic properties somewhat similar to Al(3+) (such as Fe(3+)) are somewhat unique in their ability to interact strongly with binding sites on fulvic acids.     

Catalysis of phosphoryl group transfer. The role of divalent metal ions in the hydrolysis of lactic acid O-phenyl phosphate and salicylic acid O-aryl phosphates.

The spontaneous hydrolyses of lactic acid O-phenyl phosphate (I) and, to a lesser extent, 3-hydroxybutyric acid O-phenyl phosphate (II) have been investigated and compared with similar intramolecular and bimolecular reactions. Compared to bimolecular nucleophilic reactions, the reactivity of II is similar to other systems involving the formation of a six-membered ring intermediate, which suggests that the electrostatic barrier to attack of an anionic nucleophile on a phosphate diester anion is fully present in II. The reactivity of I, as compared to that of II, would suggest that at least a partial overcoming of the electrostatic barrier takes place upon closer approimation of the two reacting centers. The Mn-2+-catalyzed hydrolysis of I exhibits saturation kinetics, consistent with the enhanced reactivity of the metal ion-substrate complex. The binding constant for this complex, determined from kinetics, is in good agreement with that obtained by electron spin resonance (ESR) titration. It is argued that the complex of Mn-2+ with II, as observed by pulsed Fourier transform nuclear magnetic resonance (NMR) techniques, is a precursor to the complex of catalytic significance. The hydrolysis of I as catalyzed by a variety of divalent metal ions suggests an optimal metal ion size. The spontaneous and metal ion catalyzed hydrolyses of salicyclic acid O-aryl phosphates (IIIa-d) proceed through cyclic acyl phosphate intermediates after expulsion of phenol. Product studies on the parent compound have failed to detect phenyl phosphate as a product in either the spontaneous or metal ion catalyzed process. The dependence of the second-order rate constant for the metal-catalyzed hydrolysis on leaving group pKa, beta-1-g, decreases significantly relative to beta-1-g for the spontaneous hydrolysis. From the collective data a specific interation of the metal ion with a pentacovalent intermediate is inferred in the rate-determining step for esters I and III. The probable consequences of these mechanistic postulates for phosphoryl transfer reactions in biological systems are discussed.     

壳聚糖-g-N.羧甲基-二(2-苯并眯唑)-1,2-乙二醇的制备及其性能

以2-溴乙酸、壳聚糖、二（2-苯并咪唑）-1,2-乙二醇为原料,利用接枝作用将化学修饰后的小分子药物二（2-苯并咪唑）-1,2-乙二醇连接在天然高分子壳聚糖（CTS）上。并以。HNMR,IR,热分析及XRD等方法对其结构进行表征并研究接枝聚合物的理化性质。本文采用络合滴定法测定了接枝聚合物对一系列重金属离子的吸附作用;采用震荡法进行悬菌定量杀菌实验;还以经典的静态失重法研究了合成的聚合物在腐蚀介质中对N80钢片腐蚀的抑制作用。结果表明：小分子药物-（2-苯并咪唑）-1,2-乙二醇在接枝到天然高分子壳聚糖后热稳定性提高,在酸中具有良好的溶解度,对金属离子吸附能力在一个较宽温度范围内得以保持;同时增强了抑菌力,降低了最小抑菌浓度;利用BBIE与CTS韵协同作用提高了聚合物对金属腐蚀的抑制能力。     

The quorum-quenching metallo-gamma-lactonase from Bacillus thuringiensis exhibits a leaving group thio effect

Lactone-hydrolyzing enzymes derived from some Bacillus species are capable of disrupting quorum sensing in bacteria that use N-acyl-l-homoserine lactones (AHLs) as intercellular signaling molecules. Despite the promise of these quorum-quenching enzymes as therapeutic and anti-biofouling agents, the ring opening mechanism and the role of metal ions in catalysis have not been elucidated. Labeling studies using (18)O, (2)H, and the AHL lactonase from Bacillus thuringiensis implicate an addition-elimination pathway for ring opening in which a solvent-derived oxygen is incorporated into the product carboxylate, identifying the alcohol as the leaving group. (1)H NMR is used to show that metal binding is required to maintain proper folding. A thio effect is measured for hydrolysis of N-hexanoyl-l-homoserine lactone and the corresponding thiolactone by AHL lactonase disubstituted with alternative metal ions, including Mn(2+), Co(2+), Zn(2+), and Cd(2+). The magnitude of the thio effect on k(cat) values and the thiophilicity of the metal ion substitutions vary in parallel and are consistent with a kinetically significant interaction between the leaving group and the active site metal center during turnover. X-ray absorption spectroscopy confirms that dicobalt substitution does not result in large structural perturbations at the active site. Finally, substitution of the dinuclear metal site with Cd(2+) results in a greatly enhanced catalyst that can hydrolyze AHLs 1600-24000-fold faster than other reported quorum-quenching enzymes.