Modulation of linoleic acid-binding properties of human serum albumin by divalent metal cations

Human serum albumin (HSA) is an abundant multiligand carrier protein, linked to progression of Alzheimer’s disease (AD). Blood HSA serves as a depot of amyloid β (Aβ) peptide. Aβ peptide-buffering properties of HSA depend on interaction with its ligands. Some of the ligands, namely, linoleic acid (LA), zinc and copper ions are involved into AD progression. To clarify the interplay between LA and metal ion binding to HSA, the dependence of LA binding to HSA on Zn²⁺, Cu²⁺, Mg²⁺ and Ca²⁺ levels and structural consequences of these interactions have been explored. Seven LA molecules are bound per HSA molecule in the absence of the metal ions. Zn²⁺ binding to HSA causes a loss of one bound LA molecule, while the other metals studied exert an opposite effect (1–2 extra LA molecules are bound). In most cases, the observed effects are not related to the metal-induced changes in HSA quaternary structure. However, the Zn²⁺-induced decline in LA capacity of HSA could be due to accumulation of multimeric HSA forms. Opposite to Ca²⁺/Mg²⁺-binding, Zn²⁺ or Cu²⁺ association with HSA induces marked changes in its hydrophobic surface. Overall, the divalent metal ions modulate LA capacity and affinity of HSA to a different extent. LA- and Ca²⁺-binding to HSA synergistically support each other. Zn²⁺ and Cu²⁺ induce more pronounced changes in hydrophobic surface and quaternary structure of HSA and its LA capacity. A misbalanced metabolism of these ions in AD could modify interactions of HSA with LA, other fatty acids and hydrophobic substances, associated with AD.     

A new principle for activity measurement of ADP or ATP dependent enzymes: fluorescence quenching of epsilon-ADP and epsilon-ATP by divalent metal ions.

The divalent metal ions Cu²⁺, Co²⁺, Mn²⁺, and Zn²⁺ form complexes with the fluorescent etheno analogs of the adenine nucleotides. The fluorescence intensity is thereby diminished. The binding strength of the metals to etheno-adenosine triphosphate is higher than to etheno-adenosine di- and monophosphate. The quenching effect of the divalent metal ions can be exploited as a simple routine activity measurement for various kinases and phosphohydrolases.     

Effects of divalent metal cations on lovastatin biosynthesis from <Emphasis Type="Italic">Aspergillus terreus</Emphasis> in chemically defined medium

Effects of six divalent metal cations: Fe²⁺, Ca²⁺, Zn²⁺, Mg²⁺, Cu²⁺and Mn²⁺ on fungal cell growth and lovastatin biosynthesis were investigated by submerged cultivation of Aspergillus terreus in a modified chemically defined medium. The influences of different initial concentrations of the above six metal cations were also examined at 1, 2, and 5 mM, respectively. Cu²⁺ apparently inhibited the cell growth, but had no influence on biosynthesis of lovastatin. All of Fe²⁺, Ca²⁺, Zn²⁺, Mg²⁺ and Mn²⁺ promoted the cell growth and lovastatin biosynthesis in different extents. The highest biomass of 13.8 ± 0.5 g l⁻¹ and specific lovastatin titres of 49.2 ± 1.4 mg gDCW⁻¹ were obtained at the level of 2 and 5 mM in the presence of Zn²⁺, respectively. The values were improved double and 14.4-fold. Excess Zn²⁺ inhibited the cell growth, but enhanced lovastatin biosynthesis with an increment of 17.6 mg l⁻¹ per mM. The interactions of all metal cations slightly inhibited the lovastatin production comparing with the existence of Zn²⁺, Fe²⁺ and Mg²⁺ solely, yet remarkably improved the cell growth. These results suggest that the divalent metal ions Zn²⁺ or Fe²⁺ influence the production by regulating the action of key enzymes such as LovD or LovF in lovastatin biosynthesis.     

Evidence for the Cd<Superscript>2+</Superscript> Activation of the Aryl Sulfatase from <Emphasis Type="Italic">Helix pomatia</Emphasis>

Often used to remove sulfate groups from carbohydrates, the regulatory properties of the aryl sulfatase from Helix pomatia remain little characterized. As many hydrolytic enzymes utilize exogenous metal ions in catalysis, the effect of various divalent metal ions on the sulfatase was investigated. Evidence for metal ion activation was collected, with Cd²⁺ being notable for effective activation. The enzyme was inhibited by Cu²⁺. The response of other common hydrolases to divalent metal ions was characterized. Activation by Cd²⁺ was not observed for chymotrypsin, rabbit liver esterase, or β-galactosidase. Instead, Cd was found to inhibit both the esterase and the galactosidase. Inhibition by Cu²⁺ and Zn²⁺ was also observed for some of these hydrolases.     

Pulsed electron spin resonance resolves the coordination site of Cu²(+) ions in α1-glycine receptor

Herein, we identify the coordination environment of Cu²⁺ in the human α1-glycine receptor (GlyR). GlyRs are members of the pentameric ligand-gated ion channel superfamily (pLGIC) that mediate fast signaling at synapses. Metal ions like Zn²⁺ and Cu²⁺ significantly modulate the activity of pLGICs, and metal ion coordination is essential for proper physiological postsynaptic inhibition by GlyR in vivo. Zn²⁺ can either potentiate or inhibit GlyR activity depending on its concentration, while Cu²⁺ is inhibitory. To better understand the molecular basis of the inhibitory effect we have used electron spin resonance to directly examine Cu²⁺ coordination and stoichiometry. We show that Cu²⁺ has one binding site per α1 subunit, and that five Cu²⁺ can be coordinated per GlyR. Cu²⁺ binds to E192 and H215 in each subunit of GlyR with a 40 μM apparent dissociation constant, consistent with earlier functional measurements. However, the coordination site does not include several residues of the agonist/antagonist binding site that were previously suggested to have roles in Cu²⁺ coordination by functional measurements. Intriguingly, the E192/H215 site has been proposed as the potentiating Zn²⁺ site. The opposing modulatory actions of these cations at a shared binding site highlight the sensitive allosteric nature of GlyR.     

Interaction of Divalent Metal Ions with Zn2+-Glycerophosphocholine Cholinephosphodiesterase from Ox Brain

The effect of divalent metal ions on the activity of glycerophosphocholine cholinephosphodiesterse from ox brain was examined. Zn²⁺- and Co²⁺-glycerophosphocholine cholinephosphodiesterases were prepared from the exposure of apoenzyme to Zn²⁺ and Co²⁺, respectively, and the properties of two metallo-phosphodiesterases were compared to those of native phosphodiesterase. Although two metallo-enzymes were similar in expressing Km value, optimum pH or sensitivity to Cu²⁺, they differed in the susceptibility to the inhibition by thiocholine or tellurite; while Co²⁺-phosphodiesterase was more sensitive to tellurites, Zn²⁺-phosphodiesterase was more susceptible to inhibition by thiocholine. In addition, Zn²⁺-phosphodiesterase was more thermo-stable than Co²⁺ enzyme. Separately, when properties of native phosphodiesterase were compared to those of each metallo-phosphodiesterase, native phosphodiesterase was found to be quite similar to Zn²⁺-phosphodiesterase in many respects. Even in thermo-stability, native enzyme resembled Zn²⁺-phosphodiesterase rather than Co²⁺-enzyme. Consistent with this, the stability of native phosphodiesterase was maintained in the presence of Zn²⁺, but not Co²⁺. Mn²⁺ was also as effective as Zn²⁺ in the stabilization of the enzyme. Noteworthy, the native enzyme was found to be inhibited competitively by Cu²⁺ with a Ki value of 20 M, and its inhibitory action was antagonized effectively by Zn²⁺ or Co²⁺. Also, choline, another competitive inhibitor of the enzyme, appeared to antagonize the inhibitory action of Cu²⁺. Taken together, it is suggested that there may be multiple binding sites for divalent metal ions in the molecule of glycerophosphocholine cholinephosphodiesterase.     

Characterization of the metal ion binding site in the anti-terminator protein, HutP, of Bacillus subtilis

HutP is an RNA-binding protein that regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis, by binding to cis-acting regulatory sequences on hut mRNA. It requires L-histidine and an Mg²⁺ ion for binding to the specific sequence within the hut mRNA. In the present study, we show that several divalent cations can mediate the HutP–RNA interactions. The best divalent cations were Mn²⁺, Zn²⁺ and Cd²⁺, followed by Mg²⁺, Co²⁺ and Ni²⁺, while Cu²⁺, Yb²⁺ and Hg²⁺ were ineffective. In the HutP–RNA interactions, divalent cations cannot be replaced by monovalent cations, suggesting that a divalent metal ion is required for mediating the protein–RNA interactions. To clarify their importance, we have crystallized HutP in the presence of three different metal ions (Mg²⁺, Mn²⁺ and Ba²⁺), which revealed the importance of the metal ion binding site. Furthermore, these analyses clearly demonstrated how the metal ions cause the structural rearrangements that are required for the hut mRNA recognition.     

A micro-scale method for determining relative metal-binding affinities of proteins

This article describes a quick and easy method for determining relative binding affinities between proteins and metal ions. The method is based on separating unbound metal ions from metal ions bound to protein by ultrafiltration using microcentrifuge ultrafiltration units. Bovine serum albumin (BSA) was used as the test protein and the relative affinity towards divalent metal ions was found to be Cu²⁺>Zn²⁺>Cd²⁺>Pb²⁺>Ni²⁺>Co²⁺, which corresponds to the relative orders reported in the literature.     

Systemic lupus erythematosus: molecular cloning and analysis of 22 individual recombinant monoclonal kappa light chains specifically hydrolyzing human myelin basic protein

Antibodies hydrolyzing myelin basic protein (MBP) can play an important role in the pathogenesis of multiple sclerosis (MS) and systemic lupus erythematosus (SLE). An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with SLE was used. Small pools of phage particles displaying light chains with different affinities for MBP were isolated by affinity chromatography on MBP‐Sepharose, and the fraction eluted with 0.5 M NaCl was used for preparation of individual monoclonal light chains (MLChs, 26–27 kDa). Seventy‐two of 440 individual colonies were randomly chosen, expressed in Escherichia coli in a soluble form, and MLChs were purified by metal chelating chromatography. Twenty‐two of 72 MLChs have high affinity and efficiently hydrolyze only MBP (not other control proteins) demonstrating various pH optima in a 5.7–9.0 range and different substrate specificity in the hydrolysis of four different MBP oligopeptides. Four MLChs demonstrated serine protease‐like and three thiol protease‐like activities, while 11 MLChs were metalloproteases. The activity of three MLChs was inhibited by both phenylmethylsulfonyl fluoride (PMSF) and Ethylenediaminetetraacetic acid (EDTA), two other by EDTA and iodoacetamide, and one by PMSF, EDTA, and iodoacetamide. The ratio of relative activity in the presence of Ca²⁺, Mg²⁺_, Mn²⁺, Ni²⁺, Zn²⁺, Cu²⁺, and Co²⁺ was individual for each of 22 MLCh preparations. It is the first examples of human MLChs, which probably can possess two or even three different proteolytic activities. These observations suggest an extreme diversity of anti‐MBP abzymes in SLE patients. The immune systems of individual SLE patients can generate a variety of anti‐MBP abzymes, which can attack MBP of myelin‐proteolipid sheath of axons and play an important role in MS and SLE pathogenesis. Copyright © 2015 John Wiley & Sons, Ltd.     

Induced Secondary Structure and Polymorphism in an Intrinsically Disordered Structural Linker of the CNS: Solid-State NMR and FTIR Spectroscopy of Myelin Basic Protein Bound to Actin

The 18.5 kDa isoform of myelin basic protein (MBP) is a peripheral membrane protein that maintains the structural integrity of the myelin sheath of the central nervous system by conjoining the cytoplasmic leaflets of oligodendrocytes and by linking the myelin membrane to the underlying cytoskeleton whose assembly it strongly promotes. It is a multifunctional, intrinsically disordered protein that behaves primarily as a structural stabilizer, but with elements of a transient or induced secondary structure that represent binding sites for calmodulin or SH3-domain-containing proteins, inter alia. In this study we used solid-state NMR (SSNMR) and Fourier transform infrared (FTIR) spectroscopy to study the conformation of 18.5 kDa MBP in association with actin microfilaments and bundles. FTIR spectroscopy of fully ¹³C,¹⁵N-labeled MBP complexed with unlabeled F-actin showed induced folding of both protein partners, viz., some increase in β-sheet content in actin, and increases in both α-helix and β-sheet content in MBP, albeit with considerable extended structure remaining. Solid-state NMR spectroscopy revealed that MBP in MBP-actin assemblies is structurally heterogeneous but gains ordered secondary structure elements (both α-helical and β-sheet), particularly in the terminal fragments and in a central immunodominant epitope. The overall conformational polymorphism of MBP is consistent with its in vivo roles as both a linker (membranes and cytoskeleton) and a putative signaling hub.     

Prion metal interaction: Is prion pathogenesis a cause or a consequence of metal imbalance?

Functional role of cellular prion protein (PrPc) has been hypothesized to be in metal homeostasis and providing cells with a superoxide dismutase (SOD)-like activity to escape damage by reactive oxygen species (ROS). PrPc interacts with a range of divalent metal ions and undergoes Cu²⁺ as well as Zn²⁺-associated endocytosis, thereby maintaining homeostasis of these and other metal ions. Conformational change to a β-sheet rich, protease resistant entity, reminiscent of the disease-associated scrapie form called PrPsc, has been found to be induced by interaction of PrPc with metal ions like Cu²⁺, Zn²⁺, Mn²⁺ and Fe²⁺. This review compiles data from various experimental studies of the interaction of metals with PrPc. The effect of metal ions on the expression and conformation of the prion protein is described in detail with emphasis on their possible physiological and pathogenic role. Further, a hypothesis is presented where attainment of altered conformation by metal-bound PrPc has been viewed as a deleterious consequence of efforts made by cells to maintain metal homeostasis. Thus, PrPc presumably sacrifices itself by converting into PrPsc form in an attempt to protect cells from the toxicity of metal imbalance. Finally, possible reasons for contradictions reported in the literature on the subject are explored and experimental approaches to resolve the same are suggested.     

4,5-Bis(diphenylphosphinoyl)-1,2,3-triazole ligand: Studies on metal complex formations in liquid-liquid distribution systems

The formation reactions of hydrophobic metal complexes of divalent typical element and transition metal ions with a novel chelating ligand containing N and O donor atoms, 4,5-bis(diphenylphosphinoyl)-1,2,3-triazole (L^TH), were investigated by the liquid-liquid distribution method carried out on metal ions between chloroform and aqueous solutions. The liquid-liquid distribution reaction formulae of metal ions via the formation of hydrophobic metal complexes were revealed, along with their equilibrium constants. Three types of hydrophobic mononuclear and binuclear metal complexes distributed into chloroform solutions were found, namely, ML₂ (M = Mg²⁺, Zn²⁺, Pb²⁺; L = L^T−), ML₂(HL) (M = Cd²⁺, Mn²⁺), and M₂L₃(OH) (M = Co²⁺, Ni²⁺, Cu²⁺). Linear free energy relationships were found between the equilibrium constants of the liquid-liquid distribution reactions and the stability constants of 1:1 complexes consisting of a divalent metal ion and a glycinate. These relationships suggest the chelate formation of N,O-coordination with a heterocyclic five-membered ring in the metal complexes with L^TH.     

Interaction of divalent metal ions with the carboxyl-terminal domain of human voltage-gated proton channel Hv1

The voltage-gated proton channel Hv1 functions as a dimer, in which the intracellular C-terminal domain of the protein is responsible for the dimeric architecture and regulates proton permeability. Although it is well known that divalent metal ions have effect on the proton channel activity, the interaction of divalent metal ions with the channel in detail is not well elucidated. Herein, we investigated the interaction of divalent metal ions with the C-terminal domain of human Hv1 by CD spectra and fluorescence spectroscopy. The divalent metal ions binding induced an obvious conformational change at pH 7 and a pH-sensitive reduction of thermostability in the C-terminal domain. The interactions were further estimated by fluorescence spectroscopy experiments. There are at least two binding sites for divalent metal ions binding to the C-terminal domain of Hv1, either of which is close to His²⁴⁴ or His²⁶⁶ residue. The binding of Zn²⁺ to the two sites both enhanced the fluorescence of the protein at pH 7, whereas the binding of other divalent metal ions to the two sites all resulted fluorescence quenching. The orders of the strength of divalent metal ions binding to the two sites from strong to weak are both Co²⁺, Ca²⁺, Ni²⁺, Mg²⁺, and Mn²⁺. The strength of Ca²⁺, Co²⁺, Mg²⁺, Mn²⁺ and Ni²⁺ binding to the site close to His²⁴⁴ is stronger than that of these divalent metal ions binding to the site close to His²⁶⁶.     

Influence of divalent copper, manganese and zinc ions on fibril nucleation and elongation of the amyloid-like yeast prion determinant Sup35p-NM

There is a large body of evidence that divalent metal ions, particularly copper, might play a role in several protein folding pathologies like Alzheimer’s disease, Parkinson’s disease or the prion diseases. However, contribution of metal ions on pathogenesis and their molecular influence on the formation of amyloid structures is not clear. Therefore, the general influence of metals on the formation of amyloids is still controversially discussed. We have utilized the well established system of yeast Sup35p-NM to investigate the role of three different metal ions, Cu²⁺, Mn²⁺ and Zn²⁺, on amyloidogenesis. Recently, it has been shown that the prion determining region NM of the Saccharomyces cerevisiae prion protein Sup35p, which is responsible for the yeast prion phenotype [PSI⁺], specifically binds Cu²⁺ ions. We further characterized the affinity of NM for Cu²⁺, which were found to be comparable to that of other amyloidogenic proteins like the mammalian prion protein PrP. The specific binding sites could be located in the aminoterminal N-region which is known to initiate formation of amyloidogenic nuclei. In the presence of Cu²⁺, fibril nucleation was significantly delayed, probably due to influences of copper on the oligomeric ensemble of soluble Sup35p-NM, since Cu²⁺ altered the tertiary structure of soluble Sup35p-NM, while no influences on fibril elongation could be detected. The secondary structure of soluble or fibrous protein and the morphology of the fibrils were apparently not altered when assembled in presence of Cu²⁺. In contrast, Mn²⁺ and Zn²⁺ did not bind to Sup35p-NM and did not exhibit significant effects on the formation of NM amyloid fibrils.     

Metalloregulation of Triple Helix Formation by Control of the Loop Conformation

The flexible polypyridine ligand, 2,2′:6′,2^″-terpyridine (terpy), was built into the backbone of oligonucleotides to form DNA conjugates. The terpy unit functioned as a good loop when the conjugates formed the bimolecular triplexes with complementary oligopurine. The triplex structure was destabilized by the specific interaction with divalent transition metal ions (Cu²⁺, Zn²⁺, and Fe²⁺), in particular Cu²⁺ ions. This ion destabilized one of the triplexes by 4.2 kcalmol^?1 or made the triplex formation constant less than 1/10³ at 298 K. This result is attributed to the substantial turbulence of the terminal structure of the triplexes.     

A spectrophotometric method for the determination of zinc, copper, and cobalt ions in metalloproteins using Zincon

Zincon (2-carboxy-2′-hydroxy-5′-sulfoformazylbenzene) has long been known as an excellent colorimetric reagent for the detection of zinc and copper ions in aqueous solution. To extend the chelator’s versatility to the quantification of metal ions in metalloproteins, the spectral properties of Zincon and its complexes with Zn²⁺, Cu²⁺, and Co²⁺ were investigated in the presence of guanidine hydrochloride and urea, two common denaturants used to labilize metal ions in proteins. These studies revealed the detection of metals to be generally more sensitive with urea. In addition, pH profiles recorded for these metals indicated the optimal pH for complex formation and stability to be 9.0. As a consequence, an optimized method that allows the facile determination of Zn²⁺, Cu²⁺, and Co²⁺ with detection limits in the high nanomolar range is presented. Furthermore, a simple two-step procedure for the quantification of both Zn²⁺ and Cu²⁺ within the same sample is described. Using the prototypical Cu²⁺/Zn²⁺-protein superoxide dismutase as an example, the effectiveness of this method of dual metal quantification in metalloproteins is demonstrated. Thus, the spectrophotometric determination of metal ions with Zincon can be exploited as a rapid and inexpensive means of assessing the metal contents of zinc-, copper-, cobalt-, and zinc/copper-containing proteins.     

The Effects of Metal Ions on the Cytotoxicity and Selectivity of a Histidine-Containing Lytic Peptide

The histidine-containing peptide L5C (PAWRHAFHWAWHMLHKAA) is a histidine-rich lytic peptide. Interactions of some divalent metal ions with peptide L5C and their effects on the cell lysis activity of the peptide were studied. The presence of Cu²⁺ caused a secondary structure change (from random coil to α-helix) which resulted in the loss of cell lysis activity in peptide L5C. Binding of Zn²⁺ to peptide L5C also reduced the lytic activity of the peptide but Zn²⁺ did not affect the secondary structure of the peptides. Instead, Zn²⁺ induced peptide L5C aggregation. Unlike Zn²⁺ and Cu²⁺, Mg²⁺ had no significant effect on the activity of peptide L5C. Further experiments revealed that formed ion-peptide L5C complexes were sensitive to pH and dissociated in acidic solutions. Peptide L5C demonstrated improved pH-selectivity in the presence of trace amount of Zn²⁺. This property of histidine-containing lytic peptides can be used to improve their therapeutic effectiveness in the treatment of cancers.     

Induction of the β-form of poly(S-carboxyethyl-l-cysteine) by divalent metalchlorides

The effects of eight divalent metal ions on fully neutralized poly(S-carboxyethyl-l-cysteine) have been studied by means of circular dichroism. Four ionic species (Cd²⁺, Cu²⁺, Zn²⁺ and Ni²⁺) effectively induce the β-form, while the other four species (Co²⁺, Ba²⁺, Ca²⁺ and Mg²⁺) are not effective. Specifically, Mg(ClO₄)₂ is ineffective, even at 1.86 m. The effect of Cu²⁺ ions on the polypeptide conformation is significant at pH values other than in the neural range. Comparison of the present results with previous ones from the lower side chain homologue, poly(S-carboxymethyl-l-cysteine), shows that Cd²⁺ and Zn²⁺ ions are more effetive but Co²⁺ ions are much less effective in the polypeptide studied here. Random coils of poly(S-carboxyethyl-l-cysteine) are more soluble while the β-form is less soluble compared with the respective conformations of the lower side-chain homologue.     

Induction of the β-form of poly(S-carboxyethyl-l-cysteine) by divalent metalchlorides

The effects of eight divalent metal ions on fully neutralized poly(S-carboxyethyl-l-cysteine) have been studied by means of circular dichroism. Four ionic species (Cd²⁺, Cu²⁺, Zn²⁺ and Ni²⁺) effectively induce the β-form, while the other four species (Co²⁺, Ba²⁺, Ca²⁺ and Mg²⁺) are not effective. Specifically, Mg(ClO₄)₂ is ineffective, even at 1.86 m. The effect of Cu²⁺ ions on the polypeptide conformation is significant at pH values other than in the neural range. Comparison of the present results with previous ones from the lower side chain homologue, poly(S-carboxymethyl-l-cysteine), shows that Cd²⁺ and Zn²⁺ ions are more effetive but Co²⁺ ions are much less effective in the polypeptide studied here. Random coils of poly(S-carboxyethyl-l-cysteine) are more soluble while the β-form is less soluble compared with the respective conformations of the lower side-chain homologue.     

Interaction of metal ions with polynucleotides and related compounds. XXII. Effect of divalent metal ions on the conformational changes of polyribonucleotides

Changes in the conformation of poly(G), poly(C), poly(U), and poly(I) in the presence of divalent metal ions Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Cd²⁺, and Zn²⁺ have been measured by means of ORD and u.v. spectra. Mg²⁺ and Ca²⁺ ions stabilize helical structures of all the polynucleotides very effectively at concentrations several orders of magnitude lower than the effective concentration of Na⁺ion. Cu²⁺ and Cd²⁺ destabilize the helical structure of polynucleotides to form random coils. Zn²⁺, Ni²⁺, Co²⁺, and Mn²⁺ions do not behave in such a clear-cut manner: they selectively stabilize some ordered structures, while destabilizing others, depending on the ligand strength of the nucleotide base as well as the preferred conformation of that polynucleotide.