Investigating metal-binding in proteins by nuclear magnetic resonance

Metal ions play a key role for the function of many proteins. The interaction of the metal ion with the protein and its involvement in the function of the protein vary widely. In some proteins, the metal ion is bound tightly to the ligand residues and may be the key player in the function of the protein, as in the case of blue copper proteins. In other proteins, the metal ion is bound only temporarily and loosely to the protein, as in the case of some metalloenzymes and other proteins where the metal ion acts as a cofactor necessary for the function of the protein. Such proteins are often known as metal ion-activated proteins. The review focuses on recent nuclear magnetic resonance (NMR) studies of a series of metal-dependent proteins and the characterization of the metal-binding sites. In particular, we focus on NMR techniques for studying metal binding to proteins such as chemical shift mapping, paramagnetic NMR and changes in backbone dynamics upon metal binding. Received 12 October 2006; received after revision 30 November 2006; accepted 5 February 2007     

Metallomics and metalloproteomics

Metallomics and metalloproteomics are emerging fields addressing the role, uptake, transport and storage of trace metals essential for protein functions. The methodologies utilized in metallomics and metalloproteomics to provide information on the identity, quantity and function of metalloproteins are discussed. The most widely used approach is through inductively coupled plasma mass spectrometry to identify the metal bound to a protein, and electrospray ionization mass spectrometry to elucidate the structure, dynamics and function of a metal-protein complex. Other approaches include X-ray absorption and X-ray fluorescence spectroscopies, and bioinformatics sequence analysis. X-ray absorption spectroscopy utilizing a synchrotron radiation source is a powerful tool to provide a direct analysis of metal bound to proteins and proteomic metal distribution in biological matrices. With the advent of genome sequencing, a large database of protein primary structures has been established, and specific tools to identify metalloproteins in the genome sequences have been developed. Received 8 April 2008; received after revision 12 May 2008; accepted 15 May 2008     

Ambivalent effect of protein binding on computed distributions of metal ions complexed by ligands in blood plasma

Summary Although the absolute concentrations of metal complexes in blood plasma are controlled by protein binding, the percentage distribution of transition metal ions amongst low molecular weight ligands is not. Thus, computer simulations which omit protein equilibria can nevertheless afford reliable information about such metals in the biofluid.Acknowledgments. One of us (P. M. M.) thanks the University of Cape Town and the C. S. I. R. for financial assistance. Computations were performed on both of the University of Cape Town's UNIVAC 1106 system and the St. Andrews University's IBM 360/44. Address ofP. W. Linder: University of Cape Town, Rondebosch, Cape (South Africa).     

Gluconolactonase: a zinc containing metalloprotein

Summary A metal analysis of bovine hepatic gluconolactonase indicates the presence of at least 1 atom of tightly bound zinc per enzyme subunit in this hexameric protein. Other divalent metals are present in lesser quantities and are subject to removal by EDTA. Activation energies for the manganese and magnesium catalyzed reactions are 5.3 and 11.0 kcal/mole, respectively.     

Evolution of the arginase fold and functional diversity

Novel structural superfamilies can be identified among the large number of protein structures deposited in the Protein Data Bank based on conservation of fold in addition to conservation of amino acid sequence. Since sequence diverges more rapidly than fold in protein Evolution, proteins with little or no significant sequence identity are occasionally observed to adopt similar folds, thereby reflecting unanticipated evolutionary relationships. Here, we review the unique alpha/beta fold first observed in the manganese metalloenzyme rat liver arginase, consisting of a parallel eight-stranded beta-sheet surrounded by several helices, and its evolutionary relationship with the zinc-requiring and/or iron-requiring histone deacetylases and acetylpolyamine amidohydrolases. Structural comparisons reveal key features of the core alpha/beta fold that contribute to the divergent metal ion specificity and stoichiometry required for the chemical and biological functions of these enzymes.     

Scavenger, transducer, RNA chaperone? What ligands of the prion protein teach us about its function

Prion protein, a misfolded isoform of which is the essential component of the agent of prion diseases, still remains an enigmatic protein whose physiological functions are at best hypothetical. To gain a better insight into its putative role, many studies were undertaken to look for molecules that bind prion protein, and have notably identified divalent metal ions, several proteins, and nucleic acids. At first sight, the diversity of prion protein’s ligands seems of little help to infer a plausible function. However, the intrinsically disordered property of its N-terminal tail and the potential of the protein to adopt a transmembrane topology, can both be taken into account to predict its different states during its cellular cycle and its possible functions, of which the most promising correspond to a general scavenger, a sensor or adaptor in a signaling cascade, and an RNA chaperone. Received 16 August 2006; received after revision 7 November 2006; accepted 13 December 2006     

Metallothionein induced in the frogXenopus laevis

Summary A low molecular weight cadmium- and copper-binding protein, induced in the liver of the frog,Xenopus laevis, by the administration of cadmium, was shown to consist of a single isoprotein and was characterized as an amphibian metallothionein based on its high cysteine and metal content, its low molecular weight, and the lack of aromatic amino acids.The authors thank Dr K. Kubota for his encouragement.     

Metal accumulation inAgaricus bisporus: Influence of Cd and Cu on growth and tyrosinase activity

Summary To investigate heavy metal uptake in the common mushroomAgaricus bisporus, commercial cultures of the fungus were exposed to different amounts of copper ad cadmium. In contrast to copper, cadmium already exerted pronounced toxic effects at low concentrations (>10 M) in the compost and concomitantly enhanced the uptake of copper. Tyrosinase activity measured in the fruit bodies increased sharply, when small amounts of Cd (10 M) were added to copper-rich compost. Gel filtration experiments with crude extracts from fruit bodies demonstrated the absence of a low molecular weight metal binding protein.Acknowledgments. This work was supported by the Schweizerischer Nationalfonds grant 3.420–0.78 (to K.L.) and by Hauser Champignonkulturen AG. We are indepted to Dr D. Rast for helpful discussions.     

几种纯金属熔体原子团簇的微观热收缩现象

利用高温液态金属X射线衍射仪对纯金属In、Ga、Ge、Ag、Cu、Al、Sn等在液相线以上不同温度进行了实验研究,得到了其衍射强度、结构因子、双体分布函数、径向分布函数以及平均最近邻原子间距、原子配位数.实验结果表明,纯金属In、Ge、Ag、Cu、Al、Sn等在液相线以上较大温度范围内,原子团簇都存在微观热收缩现象,几种金属的微观热收缩都具有一个共同的特点--在整个测试温度区间内热收缩是不均匀的,每种金属在各温度区间内的热收缩程度以及特征不同.在此基础上进一步探讨了微观热收缩现象的机理以及熔体内发生的结构变化.     

土壤重金属污染及防治措施

本文主要简述了土壤中重金属污染的来源、重金属存在的不同形态及在土壤-生物系统中重金属的生态效应。根据重金属污染特点，分析了目前土壤重金属污染防治措施中的优缺点。     

Investigation of the effects of copper ions on protein aggregation using a model system

Protein aggregation is a notable feature of various human disorders, including Parkinsons disease, Alzheimers disease and many others systemic amyloidoses. An increasing number of observations in vitro suggest that transition metals are able to accelerate the aggregation process of several proteins found in pathological deposits, e.g. -synuclein, amyloid (A) peptide, ₂-microglobulin and fragments of the prion protein. Here we report the effects of metal ions on the aggregation rate of human muscle acylphosphatase, a suitable model system for aggregation studies in vitro. Among the different species tested, Cu²⁺ produced the most remarkable acceleration of aggregation, the rate of the process being 2.5-fold higher in the presence of 0.1 mM metal concentration. Data reported in the literature suggest the possible role played by histidine residues or negatively charged clusters present in the amino acid sequence in Cu²⁺-mediated aggregation of pathological proteins. Acylphosphatase does not contain histidine residues and is a basic protein. A number of histidine-containing mutational variants of acylphosphatase were produced to evaluate the importance of histidine in the aggregation process. The Cu²⁺-induced acceleration of aggregation was not significantly altered in the protein variants. The different aggregation rates shown by each variant were entirely explained by the changes of hydrophobicity or propensity to form a structure introduced by the point mutation. The effect of Cu²⁺ on acylphosphatase aggregation cannot therefore be attributed to the specific factors usually invoked in the aggregation of pathological proteins. The effect, rather, seems to be a general related to the chemistry of the polypeptide backbone and could represent an additional deleterious factor resulting from the alteration of the homeostasis of metal ions in cells.Received 4 December 2003; received after revision 7 January 2004; accepted 3 February 2004     

The molecular mechanism of zinc and cadmium stress response in plants

When plants are subjected to high metal exposure, different plant species take different strategies in response to metal-induced stress. Largely, plants can be distinguished in four groups: metal-sensitive species, metal-resistant excluder species, metal-tolerant non-hyperaccumulator species, and metal-hypertolerant hyperaccumulator species, each having different molecular mechanisms to accomplish their resistance/tolerance to metal stress or reduce the negative consequences of metal toxicity. Plant responses to heavy metals are molecularly regulated in a process called metal homeostasis, which also includes regulation of the metal-induced reactive oxygen species (ROS) signaling pathway. ROS generation and signaling plays an important duel role in heavy metal detoxification and tolerance. In this review, we will compare the different molecular mechanisms of nutritional (Zn) and non-nutritional (Cd) metal homeostasis between metal-sensitive and metal-adapted species. We will also include the role of metal-induced ROS signal transduction in this comparison, with the aim to provide a comprehensive overview on how plants cope with Zn/Cd stress at the molecular level.     

Partial purification and characterization of acid phosphatase from sporulated oocysts of Eimeria tenella

Acid phosphatase of Eimeria tenella oocysts (Peak II) was purified 77-fold with a recovery of 26% using protamine sulfate precipitation, DEAE-cellulose chromatography and Sephadex G-200 gel filtration. This enzyme occurs in multiple forms as indicated by two peaks which can be separated by DEAE-cellulose chromatography and polyacrylamide gel electrophoresis. The partially purified enzyme has optimal activity at pH 4.5. With p-nitrophenyl phosphate the Km and Vmax values for (Peak II) were 25 mM and 1.57 mumol/min/mg protein, respectively. The enzyme (Peak II) is strongly inhibited by Hg++, Cu++, iodoacetamide, fluoride and molybdate. Tartrate and other divalent metal ions have no effect on enzyme activity. The partially purified Peak II phosphatase is not a glycoprotein as it is not absorbed on concanavalin-A Sepharose and its treatment with bacterial neuraminidase does not alter its elution profile through DEAE cellulose.     

Conformational change of the extracellular parts of the CFTR protein during channel gating

Cystic fibrosis can be treated by potentiators, drugs that interact directly with the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^? channel to increase its open probability. These substances likely target key conformational changes occurring during channel opening and closing, however, the molecular bases of these conformational changes, and their susceptibility to manipulation are poorly understood. We have used patch clamp recording to identify changes in the three-dimensional organization of the extracellularly accessible parts of the CFTR protein during channel opening and closing. State-dependent formation of both disulfide bonds and Cd²⁺ bridges occurred for pairs of cysteine side-chains introduced into the extreme extracellular ends of transmembrane helices (TMs) 1, 6, and 12. Between each of these three TMs, we found that both disulfide bonds and metal bridges formed preferentially or exclusively in the closed state and that these inter-TM cross-links stabilized the closed state. These results indicate that the extracellular ends of these TMs are close together when the channel is closed and that they separate from each other when the channel opens. These findings identify for the first time key conformational changes in the extracellular parts of the CFTR protein that can potentially be manipulated to control channel activity.     

Biologische und chemische Wege zur Anreicherung von Spurenelementen

Summary Using as examples hemovanadin, the vanadium-containing blood pigment of the ascidianPhallusia mamillata, and ferritin, the protein for iron storage in mammals, pathways for the concentration of vanadium and of iron are described: These have been realizedin vitro and may occurin vivo. The uptake and concentration of heavy metals in nature seems to occur by means of complexing agents.High molecular weight, insoluble chelating substances which are able to bind metal ions, especially uranium and copper selectively, have been synthesized. Recovery of the metals is possible without destruction of the chelating groups, and, as in the case of ion-exchange resins, repeated cycles can be carried out without diminishing the metal-binding capacity.     

Aquaporins with selectivity for unconventional permeants

The aquaporin protein family generally seems to be designed for the selective passage of water or glycerol. Charged molecules, metal ions and even protons are strictly excluded. Recently, particular aquaporin isoforms were reported to conduct unconventional permeants, i.e., the unpolar gases carbon dioxide and nitric oxide, the polar gas ammonia, the oxidative oxygen species hydrogen peroxide, and the metalloids antimonite, arsenite and silicic acid. Here, we summarize the available data on permeability properties and physiological settings of these aquaporins and we analyze which structural features might be connected to permeability for non-water, non-glycerol solutes. Received 31 March 2007; received after revision 3 May 2007; accepted 23 May 2007     

Antimicrobial activity of metal derivatives of sulfonamides

Antimicorbial activities of a series of metal derivatives of some sulfa drugs were examined. Such metal derivatives showed higher antimicorbial activity than the parent sulfa drugs, and among the metals, gold derivatives are seen to be the most effective.     

Role of hsp70 in cytokine production

Interleukin-1 and tumor necrosis factor-alpha are potent, multifunctional cytokine mediators of inflammation and immune responses that are produced primarily by activated monocytes and macrophages. Three published papers by different groups have shown that heat shock and chemical stress with heavy metal salts or sulfhydryl reagents, all of which induce the expression of heat shock protein 70 (hsp70), concomitantly inhibit the production of these cytokines in human monocytes and mouse macrophages activated by lipopolysaccharide. These papers are reviewed and discussed in some detail. Other studies suggest that various anti-inflammatory drugs, including acetylsalicyclic acid, auranofin and dexamethasone, can also facilitate HSP expression in macrophages. However, while these studies are interesting, it is clear that not a great deal of work has been done and/or published in this area. Since many pharmaceutical companies are developing cytokine synthesis inhibitors as potential anti-inflammatory drugs, one aim of this article is to emphasize that understanding the molecular mechanism(s) that lead to increased HSP expression and decreased cytokine biosynthesis may assist in achieving this goal.     

Concentration and method of cadmium fixation by a marine Vibrio]

In many organisms, intracellular fixation of cadmium involves specific proteins, the metallothioneins. A similar phenomenon was demonstrated in a marine bacterium belonging to the genus Vibrio. The accumulation of the metal is a function of its concentration in the medium. The uptake is also increased by cysteine, which decreases the toxicity of the metal.