Binding of Zn(II) ions to α-lactalbumin

The binding of Zn(II) ions to human and bovine -lactalbumin has been studied by fluorescence, scanning microcalorimetry, and proteolytic digestion. The intrinsic tryptophan fluorescence spectrum of Ca(II)-loaded -lactalbumin is insensitive to Zn(II) binding to the strong cation binding sites (Zn:protein ratios up to 20), yet the thermal denaturation transition, as detected by intrinsic fluorescence, is shifted toward lower temperatures. On the other hand, low concentrations of Zn(II) ([Zn]:[protein]<1) shift heat sorption curves toward lower temperatures. It was concluded that -lactalbumin possesses several relatively strong Zn(II) binding sites, which are filled sequentially, the process being accompanied by protein aggregation. The strongest Zn(II) binding (5×10⁵ M^–1) increases its susceptibility to tryptic and chymotryptic digestion, slightly decreases its affinity for the fluorescent probe, bis-ANS, and alters its interactions with UDP-galactose. Zn(II) binding to aggregated forms of -lactalbumin increases its affinity to bis-ANS.     

pH-dependence of the specific binding of Cu(II) and Zn(II) ions to the amyloid-β peptide

Metal ions like Cu(II) and Zn(II) are accumulated in Alzheimer's disease amyloid plaques. The amyloid-β (Aβ) peptide involved in the disease interacts with these metal ions at neutral pH via ligands provided by the N-terminal histidines and the N-terminus. The present study uses high-resolution NMR spectroscopy to monitor the residue-specific interactions of Cu(II) and Zn(II) with (15)N- and (13)C,(15)N-labeled Aβ(1-40) peptides at varying pH levels. At pH 7.4 both ions bind to the specific ligands, competing with one another. At pH 5.5 Cu(II) retains its specific histidine ligands, while Zn(II) seems to lack residue-specific interactions. The low pH mimics acidosis which is linked to inflammatory processes in vivo. The results suggest that the cell toxic effects of redox active Cu(II) binding to Aβ may be reversed by the protective activity of non-redox active Zn(II) binding to the same major binding site under non-acidic conditions. Under acidic conditions, the protective effect of Zn(II) may be decreased or changed, since Zn(II) is less able to compete with Cu(II) for the specific binding site on the Aβ peptide under these conditions.     

Picomolar concentrations of free zinc(II) ions regulate receptor protein-tyrosine phosphatase β activity

As key enzymes in the regulation of biological phosphorylations, protein-tyrosine phosphatases are central to the control of cellular signaling and metabolism. Zinc(II) ions are known to inhibit these enzymes, but the physiological significance of this inhibition has remained elusive. Employing metal buffering for strict metal control and performing a kinetic analysis, we now demonstrate that zinc(II) ions are reversible inhibitors of the cytoplasmic catalytic domain of the receptor protein-tyrosine phosphatase β (also known as vascular endothelial protein-tyrosine phosphatase). The K(i)((Zn)) value is 21 ± 7 pm, 6 orders of magnitude lower than zinc inhibition reported previously for this enzyme. It exceeds the affinity of the most potent synthetic small molecule inhibitors targeting these enzymes. Inhibition is in the range of cellular zinc(II) ion concentrations, suggesting that zinc regulates this enzyme, which is involved in vascular physiology and angiogenesis. Thus, for some enzymes that are not recognized as zinc metalloenzymes, zinc binding inhibits rather than activates as in classical zinc enzymes. Activation then requires removal of the inhibitory zinc.     

The effect of some ions on the activity of β-galactosidase and the inhibitory effect of tris (hydroxymethyl) aminomethane

The stimulating effect of phosphate and the inhibitory effect of tris-HCl on the activity of β-galactosidase inEscherichia coli was studied. The phosphate anion antagonizes the inhibitory effect of chloride. Since a similar effect is displayed by sulphate and arsenate no specific “stimulating” effect of phosphate can take place. The tris cation has also an inhibitory effect which is antagonized by univalent cations (K⁺). The resulting β-galactosidase activity reflects the antagonisms between cations and anions present in the reaction medium.     

Interaction of zinc ions with d(CGCAATTGCG) in a 2.9 Å resolution X-ray structure

We have synthesized and crystallized in the presence of Zn(2+) ions the peptidyl-oligonucleotide adduct CH(3)CO-(Arg)(4)-NH-(CH(2))(6)-NH-p-d(CGCAATTGCG). This is the first structure obtained from a deoxyoligonucleotide crystallized in the presence of zinc ions. Zn ions are clearly visible in the 2.9 A resolution map. On the other hand, the peptide tail is not visible in the crystal structure as determined by X-ray diffraction. The terminal bases C1 and G10 are found in extra-helical positions. Their phosphates are ligands of a Zn(2+) ion, located in a special position of the unit cell. This ion plays an important role in the packing arrangement, since it binds four different DNA molecules. Two other Zn(2+) ions are also important for DNA packing. They interact specifically with the N7 atoms of the terminal G2 and G10 bases, but not with the internal G8. This result supports the hypothesis that transition metals do not interact with the bases of duplex DNA in the B form.     

Spin concentration measurements of high-spin (g′ = 4.3) rhombic iron(III) ions in biological samples: theory and application

Electron paramagnetic resonance (EPR) signals at g′ = 4.3 are commonly encountered in biological samples owing to mononuclear high-spin (S = 5/2) Fe³⁺ ions in sites of low symmetry. The present study was undertaken to develop the experimental method and a suitable g′ = 4.3 intensity standard and for accurately quantifying the amount of Fe³⁺ responsible for such signals. By following the work of Aasa and Vänngård (J. Magn. Reson. 19:308–315, 1975), we present equations relating the EPR intensity of S = 5/2 ions to the intensities of S = 1/2 standards more commonly employed in EPR spectrometry. Of the chelates tested, Fe³⁺–EDTA (1:3 ratio) in 1:3 glycerol/water (v/v), pH 2, was found to be an excellent standard for frozen-solution S = 5/2 samples at 77 K. The spin concentrations of Cu²⁺–EDTA and aqua VO²⁺, both S = 1/2 ions, and of Fe³⁺–transferrin, an S = 5/2 ion, were measured against this standard and found to agree within 2.2% of their known metal ion concentrations. Relative standard deviations of ±3.6, ±5.3 and ±2.9% in spin concentration were obtained for the three samples, respectively. The spin concentration determined for Fe³⁺–desferrioxamine of known Fe³⁺ concentration was anomalously low suggesting the presence of EPR-silent multimeric iron species in solution.     

Membrane interactions and the effect of metal ions of the amyloidogenic fragment Aβ(25-35) in comparison to Aβ(1-42)

Aβ(1−42) peptide, found as aggregated species in Alzheimer's disease brain, is linked to the onset of Alzheimer's disease. Many reports have linked metals to inducing Aβ aggregation and amyloid plaque formation. Aβ(25-35), a fragment from the C-terminal end of Aβ(1−42), lacks the metal coordinating sites found in the full-length peptide and is neurotoxic to cortical cortex cell cultures. We report solid-state NMR studies of Aβ(25-35) in model lipid membrane systems of anionic phospholipids and cholesterol, and compare structural changes to those of Aβ(1-42). When added after vesicle formation, Aβ(25-35) was found to interact with the lipid headgroups and slightly perturb the lipid acyl-chain region; when Aβ(25-35) was included during vesicle formation, it inserted deeper into the bilayer. While Aβ(25-35) retained the same β-sheet structure irrespective of the mode of addition, the longer Aβ(1-42) appeared to have an increase in β-sheet structure at the C-terminus when added to phospholipid liposomes after vesicle formation. Since the Aβ(25-35) fragment is also neurotoxic, the full-length peptide may have more than one pathway for toxicity.     

Calcium ions promote formation of amyloid β-peptide (1-40) oligomers causally implicated in neuronal toxicity of Alzheimer's disease

Amyloid β-peptide (Aβ) is directly linked to Alzheimer's disease (AD). In its monomeric form, Aβ aggregates to produce fibrils and a range of oligomers, the latter being the most neurotoxic. Dysregulation of Ca(2+) homeostasis in aging brains and in neurodegenerative disorders plays a crucial role in numerous processes and contributes to cell dysfunction and death. Here we postulated that calcium may enable or accelerate the aggregation of Aβ. We compared the aggregation pattern of Aβ(1-40) and that of Aβ(1-40)E22G, an amyloid peptide carrying the Arctic mutation that causes early onset of the disease. We found that in the presence of Ca(2+), Aβ(1-40) preferentially formed oligomers similar to those formed by Aβ(1-40)E22G with or without added Ca(2+), whereas in the absence of added Ca(2+) the Aβ(1-40) aggregated to form fibrils. Morphological similarities of the oligomers were confirmed by contact mode atomic force microscopy imaging. The distribution of oligomeric and fibrillar species in different samples was detected by gel electrophoresis and Western blot analysis, the results of which were further supported by thioflavin T fluorescence experiments. In the samples without Ca(2+), Fourier transform infrared spectroscopy revealed conversion of oligomers from an anti-parallel β-sheet to the parallel β-sheet conformation characteristic of fibrils. Overall, these results led us to conclude that calcium ions stimulate the formation of oligomers of Aβ(1-40), that have been implicated in the pathogenesis of AD.     

Effects of Hofmeister ions on the α-helical structure of proteins

The molecular conformation of proteins is sensitive to the nature of the aqueous environment. In particular, the presence of ions can stabilize or destabilize (denature) protein secondary structure. The underlying mechanisms of these actions are still not fully understood. Here, we combine circular dichroism (CD), single-molecule Förster resonance energy transfer, and atomistic computer simulations to elucidate salt-specific effects on the structure of three peptides with large α-helical propensity. CD indicates a complex ion-specific destabilization of the α-helix that can be rationalized by using a single salt-free computer simulation in combination with the recently introduced scheme of ion-partitioning between nonpolar and polar peptide surfaces. Simulations including salt provide a molecular underpinning of this partitioning concept. Furthermore, our single-molecule Förster resonance energy transfer measurements reveal highly compressed peptide conformations in molar concentrations of NaClO₄ in contrast to strong swelling in the presence of GdmCl. The compacted states observed in the presence of NaClO₄ originate from a tight ion-backbone network that leads to a highly heterogeneous secondary structure distribution and an overall lower α-helical content that would be estimated from CD. Thus, NaClO₄ denatures by inducing a molten globule-like structure that seems completely off-pathway between a fully folded helix and a coil state.     

The standard electrode potential (Eθ) predicts the prooxidant activity and the acute toxicity of metal ions

The standard electrode potential (E^θ) has been known for many decades to predict the toxicity of metal ions. We have compiled acute toxicity data from fifteen studies and find that the toxicity of thirty metal ions correlates with E^θ at r² = 0.868 when toxicity is expressed as log concentration of comparably effective doses. We have discovered an even stronger relationship between the prooxidant activity (PA) of metal ions and E^θ (and electronegativity, χ). Data compiled from thirty-four studies demonstrate that the PA of twenty-five metal ions correlates with E^θ at r² = 0.983 (and χ at r² = 0.968). PA was commonly measured as metal-induced peroxidation of cell membranes or accumulation of reactive oxygen species. None of the redox metals (capable of Fenton-like reactions) in our studies (i.e., Mn, Fe, Co, Ni, and Cu) was prooxidative or toxic beyond what was expected from E^θ or χ. We propose that the formation of superoxide-metal ion complexes is greater at greater E^θ or χ values and that these complexes, whether free or enzyme-bound, function in PA without redox cycling of the complexed ion.     

Application of 2,4,5-tris(2-pyridyl)imidazole as ‘turn-off’ fluorescence sensor for Cu(II) and Hg(II) ions and in vitro cell imaging

The 2,4,5-tris(2-pyridyl)imidazole ( L ) molecule has been evaluated as a probe for dual sensing of Hg²⁺ and Cu²⁺ ions in EtOH/HEPES buffer medium (5 mM, pH = 7.34, 1:1, v/v). Probe L shows a good sensitive and selective turn-off response in the presence of both Hg²⁺ and Cu²⁺ ions, which is comprehensible under long UV light. The probe can detect Cu²⁺ ion in the pH range 3–11 and Hg²⁺ ion in pH 6–8. The limit of detection for Cu²⁺ (0.77 μM) is well under the allowable limit prescribed by the United States Environmental Protection Agency. Two metal (Cu²⁺/Hg²⁺) ions are needed per L for complete fluorescence quenching. The probe shows marked reversibility on treatment with Na₂EDTA, making the protocol more economical for practical purposes. Paper strip coated with the L solution of EtOH can detect the presence of Cu²⁺ and Hg²⁺ ions in the sample using visible quenching of the fluorescence intensity. Density functional theory–time-dependent density functional theory (DFT–TDDFT) calculations support experimental observations, and d-orbitals of Cu²⁺/Hg²⁺ provide a nonradiative decay pathway. Cell imaging study using HDF and MDA-MB-231 cells also supported the viability of L in detecting Cu²⁺ and Hg²⁺ ions in living cells.     

Phospholemman (FXYD1) raises the affinity of the human α1β1 isoform of Na,K-ATPase for Na ions

The human α(1)/His(10)-β(1) isoform of the Na,K-ATPase has been expressed in Pichia pastoris, solubilized in n-dodecyl-β-maltoside, and purified by metal chelate chromatography. The α(1)β(1) complex spontaneously associates in vitro with the detergent-solubilized purified human FXYD1 (phospholemman) expressed in Escherichia coli. It has been confirmed that FXYD1 spontaneously associates in vitro with the α(1)/His(10)-β(1) complex and stabilizes it in an active mode. The functional properties of the α(1)/His(10)-β(1) and α(1)/His(10)-β(1)/FXYD1 complexes have been investigated by fluorescence methods. The electrochromic dye RH421 which monitors binding to and release of ions from the binding sites has been applied in equilibrium titration experiments to determine ion binding affinities and revealed that FXYD1 induces an ～30% increase of the Na(+)-binding affinity in both the E(1) and P-E(2) conformations. By contrast, it does not affect the affinities for K(+) and Rb(+) ions. Phosphorylation induced partial reactions of the enzyme have been studied as backdoor phosphorylation by inorganic phosphate and in kinetic experiments with caged ATP in order to evaluate the ATP-binding affinity and the time constant of the conformational transition, Na(3)E(1)-P → P-E(2)Na(3). No significant differences with or without FXYD1 could be detected. Rate constants of the conformational transitions Rb(2)E(1) → E(2)(Rb(2)) and E(2)(Rb(2)) → Na(3)E(1), investigated with fluorescein-labeled Na,K-ATPase, showed only minor or no effects of FXYD1, respectively. The conclusion from all these experiments is that FXYD1 raises the binding affinity of α(1)β(1) for Na ions, presumably at the third Na-selective binding site. In whole cell expression studies FXYD1 reduces the apparent affinity for Na ions. Possible reasons for the difference from this study using the purified recombinant Na,K-ATPase are discussed.     

Considerations on the mechanism of photosynthetic water oxidation – dual role of oxo-bridges between Mn ions in (i) redox-potential maintenance and (ii) proton abstraction from substrate water

Two mechanistic problems of photosynthetic water oxidation at the Mn complex of Photosystem II (PS II) are considered. (I) In the four Mn-oxidizing transitions, any pure Mn oxidation is predicted to cause an increase in redox potential that renders subsequent oxidation steps impossible (redox-potential problem). Formation of unprotonated oxo-bridges may counteract the potential increase. (II) The O–O formation step without any high-pK bases acting as proton acceptors is energetically unfavorable (acceptor-base problem). The pK of oxides in a bridging position between Mn ions may increase drastically upon reduction of Mn in the water-oxidation step (>10 units), thus rendering them favorable proton acceptors. It is proposed that in PS II, in the course of the four oxidizing transitions at least two unprotonated oxo-bridges are formed. Thereby (i) a redox potential increase is prevented and (ii) proton acceptors are prepared for the O–O formation step. Water oxidation in the O–O bond formation step is facilitated by simultaneous Mn reduction and proton transfer to bridging oxides amounting to hydrogen atom or hydride transfer from substrate water to the Mn-oxo core of the Mn complex of PS II.     

Effect of complex formation by taxifolin and naringenin with Cu(i) ions on the distribution of the components of complexes in the octanol–water system

The interaction of two structurally close flavanones: taxifolin and naringenin with copper(I) ions and its effect on the distribution of flavonoids and the corresponding ions in a biphasic system octanol–water have been studied. It has been shown that these polyphenols form complexes with copper ions of different stoichiometric ratio depending on the pH of medium (5.4, 7.4, and 9.0). The interaction of the flavonoids with copper ions leads to an increase in the fraction of polyphenols in the water phase at all pH values examined. The fraction of metal ions in octanol in the presence of both taxifolin and naringenin is maximal in the range of neutral pH values. The parameters obtained in the study, such as the partition coefficient and the coefficient of distribution in a biphasic system octanol–water (logP and logD) form the physicochemical basis necessary for the estimation of the bioavailability of flavonoids and the corresponding metal ions upon their combined consumption.     

Syntheses of (±)-Epoformin (Desoxyepoxydon) and (±)-Epiepoformin (Desoxyepiepoxydon)

Boron is an essential nutrient for plants, but it is toxic in excess. Transgenic rice plants expressing an Arabidopsis thaliana borate efflux transporter gene, AtBOR4, at a low level exhibited increased tolerance to excess boron. Those lines with high levels of expression exhibited reduced growth. These findings suggest a potential of the borate transporter BOR4 for the generation of high-boron tolerant rice.     

Synthesis of (S)-(—)-Vertinolide

(S)-(?)-Vertinolide 1 was synthesized via the tetronic acid derivative 6 from (S)-(?)-tetrahydro-2-methyl-5-oxo-2-furancarboxylic acid 3. (±)-Vertinolide was also synthesized from (±)-3.     

Synthesis of (±)-licarin-B

The synthesis of (±)-licarin-B, a neolignan of Licaria aritu Ducke (Lauraceae), was achieved by pyrolysis of 3-hydroxy-3-piperonyl-1-propyl-2-methoxy-4-(E)-propenylphenyl ethers.     

Synthesis of (–)-Muricatacin

The synthesis of (–)-muricatacin starting from 1-bromododecane and 2-pentyn-l-ol is described. 2-Pentadecyn-1-ol (4), which was prepared from 1-bromododecane (2) and 2-pentyn-1-ol (3), was converted to epoxy alcohol 6 through a two-step reaction sequence, 6 being successively submitted to tosylation, iodination, chain extension with tert-butyl lithioacetate, and acid-catalyzed cyclization to give (–)-muricatacin (1a). Recrystallization afforded optically pure 1a.     

Synthesis of (±)-Homosarkomycin and (±)-Rosaprostol

(±)-Homosarkomycin (2) and (±)-rosaprostol (3) were synthesized from (±)-methyl 2-oxo-bicyclo[3.1.0]hexane-1-carboxylate (1) by using the nucleophilic ring opening reaction on the double-activated cyclopropane ring as the key step.