NMR structures of paramagnetic metalloproteins

Metalloproteins represent a large share of the proteome and many of them contain paramagnetic metal ions. The knowledge, at atomic resolution, of their structure in solution is important to understand processes in which they are involved, such as electron transfer mechanisms, enzymatic reactions, metal homeostasis and metal trafficking, as well as interactions with their partners. Formerly considered as unfeasible, the first structure in solution by nuclear magnetic resonance (NMR) of a paramagnetic protein was obtained in 1994. Methodological and instrumental advancements pursued over the last decade are such that NMR structure of paramagnetic proteins may be now routinely obtained. We focus here on approaches and problems related to the structure determination of paramagnetic proteins in solution through NMR spectroscopy. After a survey of the background theory, we show how the effects produced by the presence of a paramagnetic metal ion on the NMR parameters, which are in many cases deleterious for the detection of NMR spectra, can be overcome and turned into an additional source of structural restraints. We also briefly address features and perspectives given by the use of 13C-detected protonless NMR spectroscopy for proteins in solution. The structural information obtained through the exploitation of a paramagnetic center are discussed for some Cu2+ -binding proteins and for Ca2+ -binding proteins, where the replacement of a diamagnetic metal ion with suitable paramagnetic metal ions suggests novel approaches to the structural characterization of proteins containing diamagnetic and NMR-silent metal ions.     

Purified Human Neuromelanin, Synthetic Dopamine Melanin as a Potential Model Pigment, and the Normal Human Substantia Nigra: Characterization by Electron Paramagnetic Resonance Spectroscopy

Abstract: Neuromelanin is a poorly understood pigment that accumulates in catecholaminergic neurons during normal aging. Electron paramagnetic resonance spectroscopy, an especially effective technique for investigating melanins, is used in the present study to show unambiguously that neuromelanin is a melanin; however, it is not well modeled by synthetic dopamine melanin and thus is an atypical melanin. Some of the unusual features of neuromelanin can be explained by postulating two distinct sources for its free radicals, the dominant one possibly derived from a precursor containing sulfur. Examination of human substantia nigra by electron paramagnetic resonance spectroscopy during the purification of neuromelanin also demonstrates, contrary to some other studies, that a portion of the paramagnetic metal ions in this tissue are bound to the pigment in situ. Combined with previous histochemical data, these observations have implications for the mechanism through which neuromelanin accumulates in vivo and are consistent with its having a cytoprotective function under normal conditions, but a cytotoxic role at advanced ages and in patients with Parkinson's disease. Other results of this study show that homogenizing tissues during the purification of any natural pigment may cause contamination of the pigment by extraneous metal ions and that subsequent incubation in hot acid, though most effective in removing metal ions and hydrolyzing proteins, leads to degradation of melanin. A purification procedure using incubation in acid at room temperature, however, is well suited for identifying and characterizing unknown natural pigments by electron paramagnetic resonance spectroscopy.     

Binding of platinum to human transferrin.

A complex of platinum and human transferrin has been formed by appropriately combining apotransferrin (metal free protein) and potassiumchloroplatinate (K2PtCl4). Atomic absorption spectroscopy indicated that both primary bind sites on the protein participated in the complex. Electron paramagnetic resonance (EPR) examination showed that the bound platinum was not paramagnetic, and thus it is highly probable that the Pt ion is in the +2 oxidation state. The results suggest a possible mechanism for physiological distribution of third-transition-series metals.     

Determination of the vanadium content of protein solutions by electron paramagnetic resonance spectroscopy

A rapid and precise method of determining the vanadium content of protein solutions by electron paramagnetic resonance spectroscopy is reported The method is based on the linear relationship between the signal intensity of the first-derivative electron paramagnetic resonance signal and the concentration of the VO(H₂O)₅²⁺ species formed in acid solution. Six different proteins were investigated in the concentration range 10^?3-10^?5m. A precision of ±1%, an accuracy of ±3%, and a detection limit of 25ppb for vanadium was obtained. This analytical method was developed to aid in investigations of metal binding sites in proteins by vanadyl ion (VO²⁺) electron paramagnetic resonance spectroscopy.     

Transport of copper and manganese to the xylem exudate of sunflower

Abstract Absorption of copper and manganese by sunflower roots from solution cultures of varying composition was followed by measuring the concentrations of the metals appearing in whole roots, root cell sap and xylem exudate. Total copper in the fibrous roots was linearly related to the concentration of copper in the external solution but the concentration of copper released to the xylem exudate was buffered somewhat against the changes made externally. No such buffering was observed for managenese. A copper-sensitive electrode, responsive only to free cupric ions was used in conjunction with total copper analysis by atomic absorption spectrophotometry to show that little of the copper (usually < 1%) existed as a free ion in any phase of the system. Copper in the xylem exudate may be strongly complexed. An electron paramagnetic resonance spectrum of the xylem exudate indicated that manganese probably was a free divalent ion. Calculation of the electrochemical potential gradient for free cupric ions showed that no special metabolically-linked mechanism need be postulated to account for absorption of copper (or manganese) other than that necessary to maintain the transmembrane potential.     

A high-performance liquid chromatography method for determining transition metal content in proteins

Transition metals are common components of cellular proteins and the detailed study of metalloproteins necessitates the identification and quantification of bound metal ions. Screening for metals is also an informative step in the initial characterization of the numerous unknown and unclassified proteins now coming through the proteomic pipeline. We have developed a high-performance liquid chromatography method for the quantitative determination of the most prevalent biological transition metals: manganese, iron, cobalt, nickel, copper, and zinc. The method is accurate and simple and can be adapted for automated high-throughput studies. The metal analysis involves acid hydrolysis to release the metal ions into solution, followed by ion separation on a mixed-bead ion-exchange column and absorbance detection after postcolumn derivatization with the metallochromic indicator 4-(2-pyridylazo)resorcinol. The potential interferences by common components of protein solutions were investigated. The metal content of a variety of metalloproteins was analyzed and the data were compared to data obtained from inductively coupled plasma-atomic emission spectroscopy. The sensitivity of the assay allows for the detection of 0.1-0.8 nmol, depending on the metal. The amount of protein required is governed by the size of the protein and the fraction of protein with metal bound. For routine analysis 50 microg was used but for many proteins 10 microg would be sufficient. The advantages, disadvantages, and possible applications of this method are discussed.     

Enzyme-based molecular imprinting with metals

Aromatic monomers with various functional groups were utilized in horseradish peroxidase-catalyzed polymerization reactions with metal ions Cu(II), Ni(II), and Fe(III) as imprinting templates. The approach described combines molecular imprinting with enzymatic free radical coupling. Selectivity in metal ion affinity between the various polymer products was assessed and found to depend on the metal used in the imprinting process using aniline, tyramine, and phenol as monomers. Selectivity in binding metals was found when polymers imprinted with copper, nickel, or iron were screened against the three metals, with preference for the metal used in the imprinting step. A model for the structural features of the putative imprinted polymers is proposed based on electron paramagnetic resonance, NMR, and IR analysis. Specific potential benefits to this imprinting method include reactivity with a wide range of aromatic monomers to provide more diverse options for molecular recognition with the target analyte and thus polymer products with higher selectivity, mild reaction conditions for the enzyme polymerization step to enable imprinting against labile substrates, imprinted polymeric products that contain conjugated backbones that could be suitable for electronics-based biosensor applications, and a potential for combinatorial selection to further enhance specificity.     

Prospects for in vivo NMR methods in xenobiotic research in plants

The application of non-invasive nuclear magnetic resonance (NMR) methods in xenobiotic research is reviewed in relation to: (i) the characterisation of the effects of xenobiotics on the metabolism of plants and plant cell suspensions; (ii) the direct detection of xenobiotics and their degradation products in vivo; and (iii) the spatial localisation of xenobiotics and their derivatives at the subcellular and tissue levels. Novel information has been generated by in vivo NMR studies of both agrochemicals and heavy metals, but a lack of generality in the methods makes it difficult to extrapolate from one successful application to the next. In vivo NMR spectroscopy is shown to be informative when a xenobiotic perturbs metabolic pathways that are accessible to the technique, and it is useful for probing the partitioning of paramagnetic metal ions between the cytoplasm and the vacuole. The successful application of 19F NMR to the analysis of plant tissue extracts also suggests that in vivo 19F NMR spectroscopy may have a role in biotransformation studies of fluorinated xenobiotics. In contrast NMR imaging techniques have been little used for xenobiotic research in plants, and while the method has been shown to be capable of monitoring the uptake and translocation of paramagnetic ions in plants, the potential use of high resolution 1H and 19F NMR imaging for mapping agrochemicals in tissues is still in its infancy.     

Complexometric titrations of protein-bound metal ions: a method for determining binding constants

A method for quantifying the affinity of proteins for specific metal ions has been developed. Both the stoichiometry and the binding constants of the protein-bound metal ion can be determined by titrating protein-bound metal ions with complexometric reagents and observing electrochemically the change in free metal ion concentration. The technique is limited to cases where the affinity of the macromolecule for the metal ion is less than or similar to the affinity of the complexometric reagent for the metal ion. The method has been employed successfully in the study of both Cu(II) and Ag(I) binding to the apoprotein of bovine cuprozinc superoxide dismutase.     

Removal of heavy metals from aqueous solution using Rhizopus delemar mycelia in free and polyurethane-bound form

This study assesses the ability of mycelia of Rhizopus delemar (both free and immobilized on polyurethane foam) to remove heavy metals from single-ion solutions as well as from a mixture of them. All experiments were conducted using 0.5-5 mm solutions of CuSO4 x 5H2O, CoCl2-6H2O and FeSO4 7H2O. Mycelia immobilized on polyurethane foam cells showed some times increase in uptake compared with that of free cells. Metal ions accumulation from a mixed solution was decreased slightly for cobalt and iron and considerable for copper ions. Heavy metal uptake was examined in the immobilized column experiments and more than 92% heavy metal removal (mg heavy metals removed/mg heavy metals added) from a mixed solution was achieved during the 5 cycles. During these experiments, the dry weight of the immobilized cells was decreased by only 2%. These results showed that immobilized mycelia of Rhizopus delemar can be used repeatedly for removal of heavy metals from aqueous solutions.     

Metal interactions with beef heart mitochondrial ATPase

Atomic absorption and electron paramagnetic resonance spectroscopy were used to study the metal binding sites of beef heart mitochondrial ATPase (F1). Quantitative and qualitative properties of these sites are described. Two different separation techniques were able to distinguish two very tight sites from one tight (easily exchangeable) metal binding site on F1. Of these sites, two are specific for magnesium while one can be substituted with Mn2+, Co2+, or Zn2+. When MgAMP-PNP was incubated with F1, a fourth metal was bound to the enzyme. The carboxyl group modified by dicyclohexylcarbodiimide is shown not to be involved in binding of any of the tightly bound metals. Qualitative properties of the metal binding sites using the Mn2+-enzyme complex as a probe were ascertained using EPR at pH 6.8 and 8.0. CrATP and Mn2+ appear to bind to different metal sites on F1. The possible role of the metals in regulation of catalysis, and their relation to nucleotide binding is discussed.     

Studies of the interaction of potassium(I), calcium(II), magnesium(II), and copper(II) with cyclosporin A

Metal ion binding properties of the immunosuppressant drug cyclosporin A have been investigated. Complexation studies in acetonitrile solution using 1H NMR and CD spectroscopy yielded 1:1 metal-peptide binding constants (log(10)K) for potassium(I), <1, magnesium(II), 4.8+/-0.2, and calcium(II), 5.0+/-1.0. The interaction of copper(II) with cyclosporin A in methanol was investigated with UV/visible and electron paramagnetic resonance (EPR) spectroscopy. No complexation of copper(II) was observed in neutral solution. In the presence of base, monomeric copper(II) complexes were detected. These results support the possibility that cyclosporin A has ionophoric properties for biologically important essential metal ions.     

Physicochemical characterization, and relaxometry studies of micro-graphite oxide, graphene nanoplatelets, and nanoribbons

The chemistry of high-performance magnetic resonance imaging contrast agents remains an active area of research. In this work, we demonstrate that the potassium permanganate-based oxidative chemical procedures used to synthesize graphite oxide or graphene nanoparticles leads to the confinement (intercalation) of trace amounts of Mn(2+) ions between the graphene sheets, and that these manganese intercalated graphitic and graphene structures show disparate structural, chemical and magnetic properties, and high relaxivity (up to 2 order) and distinctly different nuclear magnetic resonance dispersion profiles compared to paramagnetic chelate compounds. The results taken together with other published reports on confinement of paramagnetic metal ions within single-walled carbon nanotubes (a rolled up graphene sheet) show that confinement (encapsulation or intercalation) of paramagnetic metal ions within graphene sheets, and not the size, shape or architecture of the graphitic carbon particles is the key determinant for increasing relaxivity, and thus, identifies nano confinement of paramagnetic ions as novel general strategy to develop paramagnetic metal-ion graphitic-carbon complexes as high relaxivity MRI contrast agents.     

Speciation of trace elements in proteins in human and bovine serum by size exclusion chromatography and inductively coupled plasma-mass spectrometry with a magnetic sector mass spectrometer

 Proteins are separated by size exclusion chromatography while atomic ions from the inorganic elements are detected on-line by inductively coupled plasma-mass spectrometry. A double focusing mass analyzer provides very high sensitivity, low background, and sufficient spectral resolution to separate the atomic ions of interest from most polyatomic ions at the same nominal m/z value. The chromatograms show the distribution of the elements of interest between protein-bound and free fractions and provide the approximate molecular weights of those protein fractions that contain the elements monitored. The distribution of various elements, including V, Mo, Fe, Co, Mn, and lanthanides, in human or bovine serum samples are shown. Alkali metals and Tl are present primarily as free metal ions and are not bound to proteins. Inorganic elements spiked into the serum samples can be followed into various proteins. EDTA does not remove Fe, Pb, Sn, or Th from the proteins but does extract Mn from some proteins. Procedures for determining the effects of breaking disulfide linkages on the metal binding characteristics of proteins are also described. Received: 22 March 1999 / Accepted: 16 June 1999     

1H NMR techniques in studies of transport of paramagnetic ions in multicellular systems

Two different pulse sequences used in 1H NMR spectroscopy termed free induction decay amplitude recovery (FIDAR) and spin-echo recovery (SER) were applied to studies of transport of paramagnetic ions in multicellular systems. The molar relaxivity of several paramagnetic species (Fe3+, Co2+, Ni2+, Cu2+, Mn2+, MnEDTA2-, dextran-magnetite) in water solutions was measured at 32 MHz resonance frequency. Ionic transport was studied using Mn2+ and MnEDTA2- as models for cations and anions, respectively, and plant root tissue as a model of a multicellular system.     

EPR spectroscopic analysis of U7 hammerhead ribozyme dynamics during metal ion induced folding

Electron paramagnetic resonance (EPR) spectroscopy was used to examine changes in internal structure and dynamics of the hammerhead ribozyme upon metal ion induced folding, changes in pH, and the presence and absence of ribozyme inhibitors. A nitroxide spin-label was attached to nucleotide U7 of the HH16 catalytic core, and this modified ribozyme was observed to retain catalytic activity. U7 was shown by EPR spectroscopy to be more mobile in the ribozyme-product complex than in either the unfolded ribozyme or the ribozyme-substrate complex. A two-step divalent metal ion dependent folding pathway was observed for the ribozyme-substrate complex with a weak first transition observed at 0.25 mM Mg2+ and a strong second transition observed around 10 mM Mg2+, in agreement with studies using other biophysical and biochemical techniques. Previously, ribozyme activity was observed in the absence of divalent metal ions and the presence of high concentrations of monovalent metal ions, although the activity was less than that observed in the presence of divalent metal ions. Here, we observed similar dynamics for U7 in the presence of 4 M Na+ or Li+, which were distinctively different than that observed in the presence of 10 mM Mg2+, indicating that U7 of the catalytic core forms a different microenvironment under monovalent versus divalent metal ion conditions. Interestingly, the catalytically efficient microenvironment of U7 was similar to that observed in a solution containing 1 M Na+ upon addition of one divalent metal ion per ribozyme. In summary, these results demonstrate that changes in local dynamics, as detected by EPR spectroscopy, can be used to study conformational changes associated with RNA folding and function.     

Three-dimensional structure of human [113Cd7]metallothionein-2 in solution determined by nuclear magnetic resonance spectroscopy

The three-dimensional structure of human [113Cd7]metallothionein-2 was determined by nuclear magnetic resonance spectroscopy in solution. Sequence-specific 1H resonance assignments were obtained using the sequential assignment method. The input for the structure calculations consisted of the metal-cysteine co-ordinative bonds identified with heteronuclear correlation spectroscopy, 1H-1H distance constraints from nuclear Overhauser enhancement spectroscopy, and spin-spin coupling constants 3JHN alpha and 3J alpha beta. The molecule consists of two domains, the beta-domain including amino acid residues 1 to 30 and three metal ions, and the alpha-domain including residues 31 to 61 and four metal ions. The nuclear magnetic resonance data present no evidence for a preferred relative orientation of the two domains. The polypeptide-to-metal co-ordinative bonds in human metallothionein-2 are identical to those in the previously determined solution structures of rat metallothionein-2 and rabbit metallothionein-2a, and the polypeptide conformations in the three proteins are also closely similar.     

Characterization of a small metal binding protein from Nitrosomonas europaea

A small metal-binding protein (SmbP) with no known similarity to other proteins in current databases was isolated and characterized from the periplasm of Nitrosomonas europaea. The primary structure of this small (9.9 kDa) monomeric protein is characterized by a series of 10 repeats of a seven amino acid motif and an unusually high number of histidine residues. The protein was isolated from N. europaea with Cu(II) bound but was found to be capable of binding multiple equivalents of a variety of divalent and trivalent metals. The protein was overexpressed in Escherichia coli and used for the study of its metal-binding properties by UV/vis, circular dichroism (CD), and electron paramagnetic resonance (EPR) spectroscopy and equilibrium dialysis and isothermal titration calorimetry. The protein was found to bind up to six Cu(II) atoms with dissociation constants of approximately 0.1 microM for the first two metal ions and approximately 10 microM for the next four. Binding of Cu(II) resulted in spectroscopic features illustrating two distinctive geometries, as determined by EPR spectroscopy. The levels of SmbP in the periplasm were found to increase by increasing the levels of copper in the growth media. This protein is proposed to have a role in cellular copper management in the ammonia-oxidizing bacterium N. europaea.     

Paramagnetic relaxation as a tool for solution structure determination: Clostridium pasteurianum ferredoxin as an example

The possibility of using the relaxation properties of nuclei for solution structure determination of paramagnetic metalloproteins is critically evaluated. First of all, it is theoretically and experimentally demonstrated that magnetization recovery in non-selective inversion recovery experiments can be approximated to an exponential in both diamagnetic and paramagnetic systems. This permits the estimate of the contribution of paramagnetic relaxation when dominant or sizable. Then, it is shown that the averaging of paramagnetic relaxation rates due to cross relaxation is often tolerably small with respect to the use of paramagnetic relaxation rates as constraints for structural determination. Finally, a protocol is proposed to use such paramagnetic relaxation rates, which depend on the sixth power of the metal to resonating nucleus distance, as constraints for solution structure determination of proteins. As an example, the available solution structure of the oxidized ferredoxin from Clostridium pasteurianum has been significantly improved in resolution especially in the proximity of the metal ions by using 69 new constraints based on paramagnetic relaxation. Proteins 29:348–358, 1997. © 1997 Wiley-Liss, Inc.     

Iron-depleted reaction centers from Rhodopseudomonas sphaeroides R-26.1: characterization and reconstitution with Fe2+, Mn2+, Co2+, Ni2+, Cu2+, and Zn2+

Reaction centers (RCs) from the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26.1 were depleted of Fe by a simple procedure involving reversible dissociation of the H subunit. The resulting intact Fe-depleted RCs contained 0.1-0.2 Fe per RC as determined from atomic absorption and electron paramagnetic resonance (EPR) spectroscopy. Fe-depleted RCs that have no metal ion occupying the Fe site differed from native RCs in the following respects: (1) the rate of electron transfer from QA- to QB exhibited nonexponential kinetics with the majority of RCs having a rate constant slower by only a factor of approximately 2, (2) the efficiency of light-induced charge separation (DQA----D+QA-) produced by a saturating flash decreased to 63%, and (3) QA appeared readily reducible to QA2-. Various divalent metal ions were subsequently incorporated into the Fe site. The electron transfer characteristics of Fe-depleted RCs reconstituted with Fe2+, Mn2+, Co2+, Ni2+, Cu2+, and Zn2+ were essentially the same as those of native RCs. These results demonstrate that neither Fe2+ nor any divalent metal ion is required for rapid electron transfer from QA- to QB. However, the presence of a metal ion in the Fe site is necessary to establish the characteristic, native, electron-transfer properties of QA. The lack of a dominant role of Fe2+ or other divalent metals in the observed rate of electron transfer from QA- to QB suggests that a rate-limiting step (for example, a protonation event or a light-induced structural change) precedes electron transfer.