Gd<Superscript>3+</Superscript>-chelated lipid accelerates solid-state NMR spectroscopy of seven-transmembrane proteins

Solid-state NMR (SSNMR) is an attractive technique for studying large membrane proteins in membrane-mimetic environments. However, SSNMR experiments often suffer from low efficiency, due to the inherent low sensitivity and the long recycle delays needed to recover the magnetization. Here we demonstrate that the incorporation of a small amount of a Gd³⁺-chelated lipid, Gd³⁺-DMPE-DTPA, into proteoliposomes greatly shortens the spin–lattice relaxation time (¹H-T ₁) of lipid-reconstituted membrane proteins and accelerates the data collection. This effect has been evaluated on a 30 kDa, seven-transmembrane protein, Leptosphaeria rhodopsin. With the Gd³⁺-chelated lipid, we can perform 2D SSNMR experiments 3 times faster than by diamagnetic control. By combining this paramagnetic relaxation-assisted data collection with non-uniform sampling, the 3D experimental times are reduced eightfold with respect to traditional 3D experiments on diamagnetic samples. A comparison between the paramagnetic relaxation enhancement (PRE) effects of Cu²⁺- and Gd³⁺-chelated lipids indicates the much higher relaxivity of the latter. Hence, a tenfold lower concentration is needed for Gd³⁺-chelated lipids to achieve comparable PRE effects to Cu²⁺-chelated lipids. In addition, Gd³⁺-chelated lipids neither alter the protein structures nor induce significant line-width broadening of the protein signals. This work is expected to be beneficial for structural and dynamic studies of large membrane proteins by SSNMR.     

Determination of the mode of interaction of Mn2+ and Cu2+ with cis-inositol by 13C NMR spectroscopy

Natural abundance ¹³C nuclear magnetic resonance spectroscopy (¹³C NMR) was used to study the mode of binding of Mn²⁺ and Cu²⁺ to the cyclitol, cis-inositol. Resonance linewidths and the electron nuclear relaxation rates [(T₁^e)^?1 values] were used to establish that a unique binding site exists for these metal-ions on this cyclitol involving only the three axial hydroxyl groups. This work may aid in the development of new organometallic complexes used as paramagnetic relaxation agents in magnetic resonance imaging research.     

Towards extracellular Ca<Superscript>2+</Superscript> sensing by MRI: synthesis and calcium-dependent <Superscript>1</Superscript>H and <Superscript>17</Superscript>O relaxation studies of two novel bismacrocyclic Gd<Superscript>3+</Superscript> complexes

Two new bismacrocyclic Gd³⁺ chelates containing a specific Ca²⁺ binding site were synthesized as potential MRI contrast agents for the detection of Ca²⁺ concentration changes at the millimolar level in the extracellular space. In the ligands, the Ca²⁺-sensitive BAPTA-bisamide central part is separated from the DO3A macrocycles either by an ethylene (L¹) or by a propylene (L²) unit [H₄BAPTA is 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; H₃DO₃A is 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid]. The sensitivity of the Gd³⁺ complexes towards Ca²⁺ and Mg²⁺ was studied by ¹H relaxometric titrations. A maximum relaxivity increase of 15 and 10% was observed upon Ca²⁺ binding to Gd₂L¹ and Gd₂L², respectively, with a distinct selectivity of Gd₂L¹ towards Ca²⁺ compared with Mg²⁺. For Ca²⁺ binding, association constants of log K = 1.9 (Gd₂L¹) and log K = 2.7 (Gd₂L²) were determined by relaxometry. Luminescence lifetime measurements and UV–vis spectrophotometry on the corresponding Eu³⁺ analogues proved that the complexes exist in the form of monohydrated and nonhydrated species; Ca²⁺ binding in the central part of the ligand induces the formation of the monohydrated state. The increasing hydration number accounts for the relaxivity increase observed on Ca²⁺ addition. A ¹H nuclear magnetic relaxation dispersion and ¹⁷O NMR study on Gd₂L¹ in the absence and in the presence of Ca²⁺ was performed to assess the microscopic parameters influencing relaxivity. On Ca²⁺ binding, the water exchange is slightly accelerated, which is likely related to the increased steric demand of the central part leading to a destabilization of the Ln–water binding interaction. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Synthesis and characterization of a macrocyclic Schiff base Gd complex as a relaxation agent for a faster acquisition of H NMR spectra of ethanol

Quantitative ²H NMR analysis at the natural abundance represents a well-recognized and efficient method for the identification of the origin of ethanol from different sources. An intrinsic limitation of the protocol used is the long time required, about 8 h, because of the long T₁ values of the ²H resonances. In this work we propose the use a paramagnetic relaxation agent that significantly catalyzes the relaxation times and reduces the total time of the analysis. This agent is the macrocyclic Schiff base complex [Gd(H₂L)(H₂O)₃(EtOH)](Cl)₃ · 2 EtOH (H₂L is the [1 + 1] macrocycle derived from the condensation of 3,3^′-(3-oxapentane-1,5-diyldioxy)bis(2-hydroxybenzaldehyde) with 1,5-diamino-3-azamethylpentane), which is highly stable and soluble in the alcoholic solution used. Elemental analysis, IR and mass spectrometry have characterized this complex. The homogeneity of the complex and the correct Gd:Cl=1:3 ratio was established by SEM-EDS measurements. Further characterization of the paramagnetic complex has been achieved by measuring the magnetic field dependence of the ¹H longitudinal nuclear magnetic relaxation time of a 1 mM solution in CH₃OD with a field-cycling relaxometer. The Gd^III ion accommodates up to four methanol molecules in its inner coordination sphere, whose rapid exchange with the bulk provides an efficient relaxation mechanism. The addition of about 37 mg of the complex to a solution of ethanol (3.0 g) and tetramethylurea (TMU) (1.5 g) results in the reduction of the experimental time of more than 50% with a S/N ratio compatible with that required for this application.     

A multinuclear nmr relaxation study of the interaction of divalent metal ions with l-aspartic acid

Carbon-13 spin-lattice relaxation times, T₁, have been measured for aqueous solutions of L-aspartic acid, L-alanine, O-phospho-L-serine, and 2-mercapto-L-succinic acid in the presence of the paramagnetic metal ions, Cu²⁺ and Mn²⁺, and Mg²⁺ as a diamagnetic control, at ambient temperature and neutral pH. Nitrogen-15, oxygen-17 and proton relaxation times were also obtained for L-aspartic acid and phosphorus-31 relaxation times for O-phospho-L-serine under similar conditions. The structures of these complexes in solution were determined from the various metal ion-nuclei distances calculated from the paramagaetically-induced relaxation. These results indicate that the Cu²⁺ interaction with L-aspartic acid is through α-amino and β-carboxyl groups while Mn²⁺ coordinates most strongly through α-and β-carboxyl groups, with the possibility of a weak interaction through the amino group.An examination of the coordination of these divalent metal ions to an analog of L-aspartic acid in which the β-carboxyl group is replaced by a phosphate group (O-phospho-L-serine) indicated that Cu²⁺ coordination is now probably through the α-amino and phosphate groups, while this analog is a monodentate ligand for Mn²⁺ coordinating through the phosphate group. Removal of the β-carboxyl group (L-alanine) also results in Cu²⁺ coordination through the α-carboxyl and α-amino groups, and the same ligand interactions are observed with Mn²⁺. Replacement of the α-amino group of L-aspartic acid with an - SH group (2-mercapto-L-succinate) is sufficient to eliminate any specific coordination with either Cu²⁺ or Mn²⁺.     

Capturing a Reactive State of Amyloid Aggregates: NMR-BASED CHARACTERIZATION OF COPPER-BOUND ALZHEIMER DISEASE AMYLOID β-FIBRILS IN A REDOX CYCLE*

The interaction of redox-active copper ions with misfolded amyloid β (Aβ) is linked to production of reactive oxygen species (ROS), which has been associated with oxidative stress and neuronal damages in Alzheimer disease. Despite intensive studies, it is still not conclusive how the interaction of Cu⁺/Cu²⁺ with Aβ aggregates leads to ROS production even at the in vitro level. In this study, we examined the interaction between Cu⁺/Cu²⁺ and Aβ fibrils by solid-state NMR (SSNMR) and other spectroscopic methods. Our photometric studies confirmed the production of ∼60 μm hydrogen peroxide (H₂O₂) from a solution of 20 μm Cu²⁺ ions in complex with Aβ(1–40) in fibrils ([Cu²⁺]/[Aβ] = 0.4) within 2 h of incubation after addition of biological reducing agent ascorbate at the physiological concentration (∼1 mm). Furthermore, SSNMR ¹H T₁ measurements demonstrated that during ROS production the conversion of paramagnetic Cu²⁺ into diamagnetic Cu⁺ occurs while the reactive Cu⁺ ions remain bound to the amyloid fibrils. The results also suggest that O₂ is required for rapid recycling of Cu⁺ bound to Aβ back to Cu²⁺, which allows for continuous production of H₂O₂. Both ¹³C and ¹⁵N SSNMR results show that Cu⁺ coordinates to Aβ(1–40) fibrils primarily through the side chain N_δ of both His-13 and His-14, suggesting major rearrangements from the Cu²⁺ coordination via N_ϵ in the redox cycle. ¹³C SSNMR chemical shift analysis suggests that the overall Aβ conformations are largely unaffected by Cu⁺ binding. These results present crucial site-specific evidence of how the full-length Aβ in amyloid fibrils offers catalytic Cu⁺ centers.     

Solution Structure,Copper Binding and Backbone Dynamics of Recombinant Ber e 1–The Major Allergen from Brazil Nut

Background

The 2S albumin Ber e 1 is the major allergen in Brazil nuts. Previous findings indicated that the protein alone does not cause an allergenic response in mice, but the addition of components from a Brazil nut lipid fraction were required. Structural details of Ber e 1 may contribute to the understanding of the allergenic properties of the protein and its potential interaction partners.

Methodology/Principal Findings

The solution structure of recombinant Ber e 1 was solved using NMR spectroscopy and measurements of the protein back bone dynamics at a residue-specific level were extracted using ¹⁵N-spin relaxation. A hydrophobic cavity was identified in the structure of Ber e 1. Using the paramagnetic relaxation enhancement property of Cu²⁺ in conjunction with NMR, it was shown that Ber e 1 is able to specifically interact with the divalent copper ion and the binding site was modeled into the structure. The IgE binding region as well as the copper binding site show increased dynamics on both fast ps-ns timescale as well as slower µs-ms timescale.

Conclusions/Significance

The overall fold of Ber e 1 is similar to other 2S albumins, but the hydrophobic cavity resembles that of a homologous non-specific lipid transfer protein. Ber e 1 is the first 2S albumin shown to interact with Cu²⁺ ions. This Cu²⁺ binding has minimal effect on the electrostatic potential on the surface of the protein, but the charge distribution within the hydrophobic cavity is significantly altered. As the hydrophobic cavity is likely to be involved in a putative lipid interaction the Cu²⁺ can in turn affect the interaction that is essential to provoke an allergenic response.     

Proton relaxation rate and fluorometric studies of manganese and rare earth binding to concanavalin A

The binding of Mn²⁺, Ca²⁺, and rare earth ions to apoconcanavalin A has been studied by water proton relaxation enhancement, electron paramagnetic resonance spectroscopy, and fluorescence spectroscopy. An electron paramagnetic resonance and water proton relaxation rate study of the titration of apoconcanavalin A with Mn²⁺ gives evidence of two equivalent binding sites per monomer with K_D = 50 μm ± 4 μm. When a similar Mn²⁺ titration of apoconcanavalin A is performed in the presence of Ca²⁺ ion, very little free Mn²⁺ is detected by electron paramagnetic resonance until the two Mn²⁺ binding sites per monomer are filled. The substitution of a rare earth ion for Ca²⁺ ion in the above experiment often resulted in a slight displacement of Mn²⁺ from the transition metal site as detected by electron paramagnetic resonance. A water proton relaxation rate study of the titration of apoconcanavalin A with Gd³⁺ reflects two binding sites with a K_D = 40 μm ± 4 μm and two with a K_D = 200 μm ± 50 μm. The fluorescence emission spectrum of concanavalin A (λ_em = 340 nm) is slightly quenched by the addition of Tb³⁺ while Tb³⁺ fluorescence is greatly enhanced. A fluorometric titration of apoconcanavalin A with Tb³⁺ also reflects two sites with a K_D = 40 μm ± 15 μm and two with a K_D = 270 μm ± 50 μm.     

Cross-linking properties of alginate gels determined by using advanced NMR imaging and Cu<Superscript>2+</Superscript> as contrast agent

The entrapment of enzymes, drugs, cells or tissue fragments in alginates cross-linked with Ca²⁺ or Ba²⁺ has great potential in basic research, biotechnology and medicine. The swelling properties and, in turn, the mechanical stability are key factors in designing an optimally cross-linked hydrogel matrix. These parameters depend critically on the cross-linking process and seemingly minor modifications in manufacture have a large impact. Thus, sensitive and non-invasive tools are required to determine the spatial homogeneity and efficacy of the cross-linking process. Here, we show for alginate microcapsules (between 400 µm and 600 µm in diameter) that advanced ¹H NMR imaging, along with paramagnetic Cu²⁺ as contrast agent, can be used to validate the cross-linking process. Two- and three-dimensional images and maps of the spin-lattice relaxation time T₁ of Ba²⁺ cross-linked microcapsules exposed to external Cu²⁺ yielded qualitative as well as quantitative information about the accumulation of Cu²⁺ within and removal from microcapsules upon washing with Cu²⁺ free saline solution. The use of Cu²⁺ (having a slightly higher affinity constant to alginate than Ba²⁺) for gelling gave a complementary insight into the spatial homogeneity of the cross-linking process together with information about the mechanical stability of the microcapsules. The potential of this technique was demonstrated for alginates extracted from two different algal sources and cross-linked either externally by the conventional air-jet dropping method or internally by the "crystal gun" method.     

Evaluation of the influence of intermolecular electron-nucleus couplings and intrinsic metal binding sites on the measurement of <Superscript>15</Superscript>N longitudinal paramagnetic relaxation enhancements in proteins by solid-state NMR

Magic-angle spinning solid-state NMR measurements of ¹⁵N longitudinal paramagnetic relaxation enhancements (PREs) in ¹³C,¹⁵N-labeled proteins modified with Cu²⁺-chelating tags can yield multiple long-range electron-nucleus distance restraints up to ~20 Å (Nadaud et al. in J Am Chem Soc 131:8108–8120, 2009). Using the EDTA-Cu²⁺ K28C mutant of B1 immunoglobulin binding domain of protein G (GB1) as a model, we investigate the effects on such measurements of intermolecular electron-nucleus couplings and intrinsic metal binding sites, both of which may potentially complicate the interpretation of PRE data in terms of the intramolecular protein fold. To quantitatively assess the influence of intermolecular ¹⁵N-Cu²⁺ interactions we have determined a nearly complete set of longitudinal ¹⁵N PREs for a series of microcrystalline samples containing ~10, 15 and 25 mol percent of the ¹³C,¹⁵N-labeled EDTA-Cu²⁺-tagged protein diluted in a matrix of diamagnetic natural abundance GB1. The residual intermolecular interactions were found to be minor on the whole and account for only a fraction of the relatively small but systematic deviations observed between the experimental ¹⁵N PREs and corresponding values calculated using protein structural models for residues furthest removed from the EDTA-Cu²⁺ tag. This suggests that these deviations are also caused in part by other factors not related to the protein structure, such as the presence in the protein of intrinsic secondary sites capable of binding Cu²⁺ ions. To probe this issue we performed a Cu²⁺ titration study for K28C-EDTA GB1 monitored by 2D ¹⁵N-¹H solution-state NMR, which revealed that while for Cu²⁺:protein molar ratios of ≤ 1.0 Cu²⁺ binds primarily to the high-affinity EDTA tag, as anticipated, at even slightly super-stoichiometric ratios the Cu²⁺ ions can also associate with side-chains of aspartate and glutamate residues. This in turn is expected to lead to enhanced PREs for residues located in the vicinity of the secondary Cu²⁺ binding sites, and indeed many of these residues were ones found to display the elevated longitudinal ¹⁵N PREs in the solid phase.     

Tunable paramagnetic relaxation enhancements by [Gd(DPA)<Subscript>3</Subscript>]<Superscript>3−</Superscript> for protein structure analysis

Paramagnetic relaxation enhancements (PRE) present a powerful source of structural information in nuclear magnetic resonance (NMR) studies of proteins and protein–ligand complexes. In contrast to conventional PRE reagents that are covalently attached to the protein, the complex between gadolinium and three dipicolinic acid (DPA) molecules, [Gd(DPA)₃]^3?, can bind to proteins in a non-covalent yet site-specific manner. This offers straightforward access to PREs that can be scaled by using different ratios of [Gd(DPA)₃]^3? to protein, allowing quantitative distance measurements for nuclear spins within about 15 Å of the Gd³⁺ ion. Such data accurately define the metal position relative to the protein, greatly enhancing the interpretation of pseudocontact shifts induced by [Ln(DPA)₃]^3? complexes of paramagnetic lanthanide (Ln³⁺) ions other than gadolinium. As an example we studied the quaternary structure of the homodimeric GCN4 leucine zipper.     

Mapping Membrane Protein Backbone Dynamics: A Comparison of Site-Directed Spin Labeling with NMR N-Relaxation Measurements

The ability to detect nanosecond backbone dynamics with site-directed spin labeling (SDSL) in soluble proteins has been well established. However, for membrane proteins, the nitroxide appears to have more interactions with the protein surface, potentially hindering the sensitivity to backbone motions. To determine whether membrane protein backbone dynamics could be mapped with SDSL, a nitroxide was introduced at 55 independent sites in a model polytopic membrane protein, TM0026. Electron paramagnetic resonance spectral parameters were compared with NMR ¹⁵N-relaxation data. Sequential scans revealed backbone dynamics with the same trends observed for the R₁ relaxation rate, suggesting that nitroxide dynamics remain coupled to the backbone on membrane proteins.     

Mapping Membrane Protein Backbone Dynamics: A Comparison of Site-Directed Spin Labeling with NMR 15N-Relaxation Measurements

The ability to detect nanosecond backbone dynamics with site-directed spin labeling (SDSL) in soluble proteins has been well established. However, for membrane proteins, the nitroxide appears to have more interactions with the protein surface, potentially hindering the sensitivity to backbone motions. To determine whether membrane protein backbone dynamics could be mapped with SDSL, a nitroxide was introduced at 55 independent sites in a model polytopic membrane protein, TM0026. Electron paramagnetic resonance spectral parameters were compared with NMR ¹⁵N-relaxation data. Sequential scans revealed backbone dynamics with the same trends observed for the R₁ relaxation rate, suggesting that nitroxide dynamics remain coupled to the backbone on membrane proteins.     

Paramagnetic relaxation enhancement to improve sensitivity of fast NMR methods: application to intrinsically disordered proteins

We report enhanced sensitivity NMR measurements of intrinsically disordered proteins in the presence of paramagnetic relaxation enhancement (PRE) agents such as Ni²⁺-chelated DO2A. In proton-detected ¹H-¹⁵N SOFAST-HMQC and carbon-detected (H-flip)¹³CO-¹⁵N experiments, faster longitudinal relaxation enables the usage of even shorter interscan delays. This results in higher NMR signal intensities per units of experimental time, without adverse line broadening effects. At 40 mmol·L⁻¹ of the PRE agent, we obtain a 1.7- to 1.9-fold larger signal to noise (S/N) for the respective 2D NMR experiments. High solvent accessibility of intrinsically disordered protein (IDP) residues renders this class of proteins particularly amenable to the outlined approach.     

Insights into SOD1-linked amyotrophic lateral sclerosis from NMR studies of Ni2+- and other metal-ion-substituted wild-type copper–zinc superoxide dismutases

The dimeric Cu–Zn superoxide dismutase (SOD1) is a particularly interesting system for biological inorganic chemical studies because substitutions of the native Cu and/or Zn ions by a nonnative metal ion cause minimal structural changes and result in high enzymatic activity for those derivatives with Cu remaining in the Cu site. The pioneering NMR studies of the magnetically coupled derivative Cu₂Co₂SOD1 by Ivano Bertini and coworkers are of particular importance in this regard. In addition to Co²⁺, Ni²⁺ is a versatile metal ion for substitution into SOD1, showing very little disturbance of the structure in Cu₂Ni₂SOD1 and acting as a very good mimic of the native Cu ion in Ni₂Zn₂SOD1. The NMR studies presented here were inspired by and are indebted to Ivano Bertini’s paramagnetic NMR pursuits of metalloproteins. We report Ni²⁺ binding to apo wild-type SOD1 and a time-dependent Ni²⁺ migration from the Zn site to the Cu site, and the preparation and characterization of Ni₂Ni₂SOD1, which shows coordination properties similar to those of Cu₂Cu₂SOD1, namely, an anion-binding property different from that of the wild type and a possibly broken bridging His. Mutations in the human SOD1 gene can cause familial amyotrophic lateral sclerosis (ALS), and mutant SOD1 proteins with significantly altered metal-binding behaviors are implicated in causing the disease. We conclude by discussing the effects of the ALS mutations on the remarkable stabilities and metal-binding properties of wild-type SOD1 proteins and the implications concerning the causes of SOD1-linked ALS.     

Receptor- and store-operated mechanisms of calcium entry during the nanosecond electric pulse-induced cellular response

Nanosecond electric pulses have been shown to open nanopores in the cell plasma membrane by fluorescent imaging of calcium uptake and fluorescent dyes, including propidium (Pr) iodide and YO-PRO-1 (YP₁). Recently, we demonstrated that nsEPs also induce the phosphoinositide intracellular signaling cascade by phosphatidylinositol-4,5-bisphosphate (PIP₂) depletion resulting in physiological responses similar to those observed following stimulation of G_q11-coupled receptors. In this paper, we explore the role of receptor- and store-operated calcium entry (ROCE/SOCE) mechanisms in the observed response of cells to nsEP. We show that addition of the ROCE/SOCE and transient receptor potential channel (TRPC) blocker gadolinium (Gd³⁺, 300 μM) slows PIP2 depletion following 1 and 20 nsEP exposures. Lipid rafts, regions of the plasma membrane rich in PIP₂ and TRPC, are also disrupted by nsEP exposure suggesting that ROCE/SOCE mechanisms are likely impacted. Reducing the expression of stromal interaction molecule 1 (STIM1) protein, a key protein in ROCE and SOCE, in cells exposure to nsEP resulted in a reduction in induced intracellular calcium rise. Additionally, after exposure to 1 and 20 nsEPs (16.2 kV/cm, 5 Hz), intracellular calcium rises were significantly reduced by the addition of GD³⁺ and SKF-96365 (1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy] ethyl-1H-imidazole hydrochloride, 100 μM), a blocker of STIM1 interaction. However, using similar nsEP exposure parameters, SKF-96365 was less effective at reducing YP₁ uptake compared to Gd³⁺. Thus, it is possible that SKF-96365 could block STIM1 interactions within the cell, while Gd³⁺ could acts on TRPC/nanopores from outside of the cell. Our results present evidence of nsEP induces ROCE and SOCE mechanisms and demonstrate that YP₁ and Ca²⁺ cannot be used solely as markers of nsEP-induced nanoporation.     

Study of complex formation with 2-hydroxyiminocarboxylates: specific metal binding ability of 2-(4-methylthiazol-2-yl)-2-(hydroxyimino)acetic acid

Complex formation properties of a novel water soluble thiazolyloxime 2-(4-methylthiazol-2-yl)-2-(hydroxyimino)acetic acid (H₃L¹) with Cu²⁺ and Ni²⁺ were investigated in solution by potentiometrical and spectral (UV-Vis, EPR, NMR) methods. All Cu²⁺ and most of Ni²⁺ complex species detected in solution were found to have square-planar MN₄ core with oxime and heterocyclic nitrogen atoms which was rationalized in terms of destabilizing effect of repulsive interaction between oxygen atom of carboxylic group and nitrogen atom of thiazole ring in N,O-coordinated ligand conformation. It has been found that stability of metal complexes in a series of oxime ligands is dependent upon basicity of nitrogen atom of oxime group. The thiazolyloxime forms less stable complexes with Cu²⁺ but stronger ones with Ni²⁺ ions when compared to parent 2-(hydroxyimino)propanoic acid. The lower stability obtained for Cu²⁺ complexes was elucidated in terms of negative inductive effect of the thiazole and nitrile substituents as well as an effect of intramolecular attractive interaction between thiazolyl sulfur and oxime oxygen atoms in thiazolyloxime. In the case of Ni²⁺ the complexes formed are square-planar and it is why thiazolyl ligand is more effective in metal ion binding than simple 2-(hydroxyimino)propanoic acid forming only octahedral species. The solid state structure of the Co³⁺ complex K₃[Co(HL¹)₃]·5.5H₂O (1) was studied by X-ray analysis. The thiazolyloxime ligand is coordinated to Co³⁺ via oxime nitrogen and carboxylate oxygen atoms forming five-membered chelate rings.     

ESR spectroscopic evidence for hydration- and temperature-dependent spatial perturbations of a higher plant cell wall paramagnetic ion lattice

To study the influence of cell wall polyuronide structure on bound paramagnetic ion interactions, spin-spin coupling measurements were made on intact cell walls exchanged with a wide range fo Mn²⁺ and Cu²⁺ concentrations. These experiments were performed so that dimer-only intercationic nearest neighbor distances (d) and lattice constants (κ) could be calculated from the linewidth-concentration dependency. d values were estimated to be 12 and 14 Å for Cu²⁺ and Mn²⁺, respectively. At the maximal bound ion concentration, κ was 2.3–2.6, indicating that about 5–7 paramagnetic ion nearest neighbor spin-spin interactions occur per dipole in the nearly filled lattice. This latter observation strongly argues for the egg-box model of the cell wall-polyuronide lattice structure. Mn²⁺ linewidths of hydrated cell wall-bound paramagnetic ions displayed an unusual temperature dependency, whereby linewidths increased between 20°C and the temperature at which maximal linewidths were observed (T_max). T_max was inversely proportional to the degree of lattice hydration, indicating that the temperature dependency was not associated with the freezing of bound water. The relative change in Mn²⁺ linewidths, between 20°C and T_max, was affected by binding site-associated ¹H spin-lattice relaxation times, indicating that the temperature dependency is at least partially controlled by cell wall polyuronide structure.     

Reduction of blood pressure by store‐operated calcium channel blockers

The voltage‐operated Ca²⁺ channels (VOCC), which allow Ca²⁺ influx from the extracellular space, are inhibited by anti‐hypertensive agents such as verapamil and nifedipine. The Ca²⁺ entering from outside into the cell triggers Ca²⁺ release from the sarcoplasmic reticulum (SR) stores. To refill the depleted Ca²⁺ stores in the SR, another type of Ca²⁺ channels in the cell membrane, known as store‐operated Ca²⁺ channels (SOCC), are activated. These SOCCs are verapamil and nifedipine resistant, but are SKF 96465 (SK) and gadolinium (Gd³⁺) sensitive. Both SK and Gd³⁺ have been shown to reduce [Ca²⁺]_i in the smooth muscle, but their effects on blood pressure have not been reported. Our results demonstrated that both SK and Gd³⁺ produced a dose‐dependent reduction in blood pressure in rat. The combination of SK and verapamil produced an additive action in lowering the blood pressure. Furthermore, SK, but not Gd³⁺ suppressed proliferation of vascular smooth muscle cells in the absence or presence of lysophosphatidic acid (LPA). SK decreased the elevation of [Ca²⁺]_i induced by LPA, endothelin‐1 (ET‐1) and angiotensin II (Ang II), but did not affect the norepinephrine (NE)‐evoked increase in [Ca²⁺]_i. On the other hand, Gd³⁺ inhibited the LPA and Ang II induced change in [Ca²⁺]_i, but had no effect on the ET‐1 and NE induced increase in [Ca²⁺]_i. The combination of verapamil and SK abolished the LPA‐ or adenosine‐5′‐triphosphate (ATP)‐induced [Ca²⁺]_i augmentation. These results suggest that SOCC inhibitors, like VOCC blocker, may serve as promising drugs for the treatment of hypertension.     

Study of the structure of HMM · Vanadate complex

A CO₂ hydration activity for Mn(II) human carbonic anhydrase B (MnHCAB) of 7% of the activity of the native Zn²⁺ enzyme has been determined using a ¹³C magnetization—transfer NMR approach, that involves two complementary experiments. As this approach also allows a determination of the individual relaxation rates of the enzyme-bound CO₂ and HCO^?₃, an evaluation could be made of the distances between these substrates and the paramagnetic Mn²⁺ in the active site. Thus HCO^?₃ is found to bind directly to Mn²⁺, whereas CO₂ is attached relatively weakly to the enzyme without a direct bond to the metal ion.