Metal-binding properties of a single-sited transferrin fragment

A single-sited iron-binding fragment of transferrin, prepared by proteolytic cleavage with thermolysin, has been characterized. The fragment, bearing no carbohydrate, must be derived from the N-terminal half of the protein's two homologous domains. The strength of iron-binding at pH 6.7 and 7.4, and the EPR spectroscopic features of its iron and copper complexes, establish it as carying the ‘b’ site of transferrin.     

Metal-binding properties of the isolated glomerular basement membrane

The nature of binding of metal cations to the glomerular basement membrane has been investigated using isolated bovine glomerular basement membrane. Highest-affinity binding for a number of ions is attributable to the glycosaminoglycans (mostly heparan sulfate) of the membrane. Some ions, such as divalent Mn, Ca and Ni, have specific binding sites on these polymers, while for others the ion-polyelectrolyte interaction is of a non-specific nature. Both structural and binding data indicate a linear charge density of close to unity for the heparan sulfate of the glomerular basement membrane, which at the ionic composition of the plasma filtrate corresponds to a polymer surface potential of about -45 mV. Several independent observations are better explained by a model of counter-ion condensation about the glycosaminoglycans than by conventional double layer theories. These include the valence dependence of ion binding, the sharp ejection of divalent ions at a critical concentration of La3+, and the relative insensitivity of 63Ni2+ binding to NaCl concentration in the neighbourhood of physiological ionic strength. In its interactions with metal ions, the glomerular basement membrane behaves like a dilute solution of polyelectrolytes. This conclusion has important consequences for the extent of charge reduction of the filtration barrier of the kidney, bathed as it is in an electrolyte solution of mainly monovalent salts.     

Metal-binding proteins and peptides in bioremediation and phytoremediation of heavy metals

The expression of metal-binding proteins or peptides in microorganisms and plants in order to enhance heavy metal accumulation and/or tolerance has great potential. Several different peptides and proteins have been explored. This review focuses on cadmium (Cd) because of the significant importance of this metal and because of its global presence in many food materials.     

Identification of phytochelatin-related peptides in maize seedlings exposed to cadmium and obtained enzymatically in vitro

An analytical strategy based on the sensitivity of electrospray tandem mass spectrometry following a simplified and reproducible sample preparation procedure was evaluated for the determination of Cd-induced phytochelatins (PC) and related peptides in four maize varieties. In addition to the three known families of PC (PC, desGly-PC and iso-PC(Glu)) that were observed, novel PC and desGly-PC homologues lacking the N-terminal gamma-linked Glu were isolated from maize root extracts for the first time. Additionally the complete sequence of iso-PC3(Glu) was determined by tandem mass spectrometry. Peptides obtained in vivo and in vitro as the result of the reaction of glutathione with the enzyme phytochelatin synthase were compared. Minor forms detected from in vitro reactions include compounds with intramolecular or intermolecular disulfide bonds resulting from the oxidation of SH groups, phytochelatin homologues lacking the N-terminal gamma-linked Glu, and new PC-related peptides with a Cys-Cys motif. Since peptides lacking a gammaGlu residue could be generated as artifacts in electrospray mass spectrometry, the application of capillary electrophoresis with online electrospray mass spectrometry allowed the separation and detection of such peptides as endogenous molecules present in planta and as products of in vitro reactions.     

Metal-binding properties of an engineered purple CuA center in azurin

A CUA center engineered into Pseudomonas aeruginosa azurin was studied by metal substitution. Metal-binding properties were determined by electronic absorption (UV-vis) and electrospray ionization mass spectrometry (ESI-MS). The metal-binding site readily binds thiophilic metal ions, such as Hg(II), Ag(I), Cu(I), Cd(II), and Au(I). Harder metal ions, like Co(II), bind to apo-CuA-azurin only under basic conditions (pH 9.1-9.2). The results obtained from these studies indicate that two factors influence metal binding in CuA azurin: (1) the site favors metal combinations which produce an overall +3 charge, and (2) the site binds soft, thiophilic metal ions. The results demonstrate the remarkable ability of the CuA center to maintain valence delocalization of its native metal ions and to ensure redox accessibility of only one of the two redox couples (i.e., [Cu(1.5)...Cu(1.5)]<==> [Cu(I)...Cu(I)]) under physiological conditions. These findings may lead to the preparation of new metal ion derivatives and can serve as a basis for understanding this efficient electron transfer center.     

Metal-binding capacity of arbuscular mycorrhizal mycelium

Experiments with excised mycelium of several Glomus spp. with different histories of exposure to heavy metals were carried out to measure their capacities to bind Cd and Zn. Cd sorption was followed for up to 6 h of incubation to determine its time course relationships. Controls treated with a metabolic inhibitor were included to evaluate whether sorption was due to active uptake or passive adsorption. The effect of ion competition (effects of Ca or Zn on Cd sorption) and general measurements of cation exchange capacity (CEC) of roots and hyphae were also performed. The results showed that AM mycelium has a high metal sorption capacity relative to other microorganisms, and a CEC comparable to other fungi. Metal sorption was rapid (<30 min) and appeared mainly to be due to passive adsorption. Adsorption was highest in a metal-tolerant G. mosseae isolate and intermediate for a fungus isolated from a soil treated with metal-contaminated sludge. The former adsorbed up to 0.5 mg Cd per mg dry biomass, which was three times the binding capacity of non-tolerant fungi, and more than 10 times higher than reported values for, e.g., the commonly used biosorption organism Rhizopus arrhizus. The implications of these results for AM involvement in plant protection against excess heavy metal uptake are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cyanobacterial metallothionein gene expressed in Escherichia coli. Metal-binding properties of the expressed protein.

The recently isolated Synechococcus gene smtA encodes the only characterised prokaryotic protein designated to be a metallothionein (MT). To examine the metal-binding properties of its product the smtA gene was expressed in Escherichia coli as a carboxyterminal extension of glutathione-S-transferase. The pH of half dissociation of Zn, Cd and Cu ions from the expressed protein was determined to be 4.10, 3.50, 2.35, respectively, indicating a high affinity for these ions (in particular for Zn in comparison to mammalian MT). E. coli expressing this gene showed enhanced (ca. 3-fold) accumulation of Zn.     

Metal-binding sites in proteins.

A dramatic increase in the number of solved metalloprotein structures and recent breakthroughs in structural analysis have provided a sufficiently detailed understanding of the structural chemistry of some metal-binding sites to allow successful design. As a result, metal-binding site design is now one of the most powerful and promising approaches for influencing protein folding, assembly, stability and catalysis.     

Metal-binding functions and antioxidant properties in human thyroid gland under iodine deficient nodular colloidal goiter

Copper and zinc levels in the tissue of thyroid gland (TG) and their metal-binding proteins metallothioneins (MT) as well as state of the antioxidant system in persons that had no thyroid disease and patients with endemic iodine deficiency nodular colloidal goiter has been investigated. In the patients with thyroid disease, oxidative damage was indicated despite elevated levels of MT-SH and glutathione, and elevated copper and decreased zinc concentration in TG tissue. MTs partly bound the excess of copper but its concentration in the unbound to MT form was two-fold compared to the control value.     

Expression of the pea gene PSMTA in E. coli. Metal-binding properties of the expressed protein

The pea (Pisum sativum L.) gene PSMTA has an ORF encoding a predicted protein with sequence similarity to class I metallothioneins (MTS). To examine the metal-binding properties of the PSMTA protein it has been expressed in E. coli as a carboxyterminal extension of glutathione-S-transferase (GST). Metal ions were associated with the expressed protein when purified from lysates of E. coli grown in metal supplemented media. The pH of half-dissociation of Zn, Cd and Cu ions from the recombinant fusion protein was determined to be 5.35, 3.95 and 1.45 respectively, compared with equivalent estimates of 4.50, 3.00 and 1.80 for equine renal MT.     

Vestibulo-protective properties of regulatory peptides

Endogenous opioid peptides take an active part in the pathogenesis of motion sickness (MS). Some regulatory peptides (substances P, gamma and des-tyr-gamma-endorphins) manifest antiemetic properties, unlike opioid peptides and morphine. Thus, we have examined the vestibulo-protective properties of some regulatory peptides during simulation of MS in cats. Vestibulo-protective properties have been discovered in some peptides: sweet water hydra undecapeptide, substances P, gamma and des-tyr-gamma-endorphins. It has been suggested that regulatory peptides take an active part in the genesis of vestibulo-protective disorders during motion sickness.     

Novel properties of antimicrobial peptides

Endogenous peptide antibiotics are known as evolutionarily old components of innate immunity. Due to interaction with cell membrane these peptides cause permeabilization of the membrane and lysis of invading microbes. However, some studies proved that antimicrobial peptides are universal multifunctional molecules and their functions extend far beyond simple antibiotics. In this review we present an overview of the general mechanism of action of antimicrobial peptides and discuss some of their additional properties, like antitumour activity, mitogenic activity, role in signal transduction pathways and adaptive immune response.     

Comparative immunological properties of enantiomeric peptides

Summary The immunogenicity of a peptide composed of only d-amino acids is compared with that of the corresponding l-peptide enantiomer. Following three administrations of 100 g of individual peptide formulated with different adjuvants (Freund's complete adjuvant, QS21, or alum) to BALB/c mice, guinea pigs and rabbits, the l-peptide elicited strong l-peptide-specific IgG antibody responses in all formulations, whereas the d-peptide-induced d-peptide-specific IgG antibodies in the Freund's complete adjuvant and QS21 formulations, but was nonimmunogenic in the alum formulation. Mouse T-cell lines induced by the d-peptide formulated in Freund's complete adjuvant were found to express significant amounts of IL-2 when they were stimulated by the d-peptide. When an equal amount of both enantiomers was mixed and administered in Freund's complete adjuvant, only an l-peptide-specific IgG antibody response was observed. These results suggest that (i) d-peptide is immunogenic when strong adjuvant is provided; (ii) the immune system has preferential recognition of l-amino acid peptide; and (iii) the d-peptide can elicit d-peptide-specific T-cell responses.     

Membrane destabilizing properties of cell-penetrating peptides

Although cell-penetrating peptides (CPPs), also denoted protein transduction domains (PTDs), have been widely used for intracellular delivery of large and hydrophilic molecules, the mechanism of uptake is still poorly understood. In a recent live cell study of the uptake of penetratin and tryptophan-containing analogues of Tat(48-60) and oligoarginine, denoted TatP59W, TatLysP59W and R(7)W, respectively, it was found that both endocytotic and non-endocytotic uptake pathways are involved [Thoren et al., Biochem. Biophys. Res. Commun. 307 (2003) 100-107]. Non-endocytotic uptake was only observed for the arginine-rich peptides TatP59W and R(7)W. In this paper, the interactions of penetratin, R(7)W, TatP59W and TatLysP59W with phospholipid vesicles are compared in the search for an understanding of the mechanisms for cellular uptake. While R(7)W, TatP59W and TatLysP59W are found to promote vesicle fusion, indicated by mixing of membrane components, penetratin merely induces vesicle aggregation. Studies of the leakage from dye-loaded vesicles indicate that none of the peptides forms membrane pores and that vesicle fusion is not accompanied by leakage of the aqueous contents of the vesicles. These observations are important for a proper interpretation of future experiments on the interactions of these peptides with model membranes. We suggest that the discovered variations in propensity to destabilize phospholipid bilayers between the peptides investigated, in some cases sufficient to induce fusion, may be related to their different cellular uptake properties.     

Metal-binding and active-site structure of di-zinc Streptomyces griseus aminopeptidase

 Streptomyces griseus aminopeptidase has been characterized to have a dinuclear active site and to follow a dinuclear hydrolytic mechanism by means of activity assay, optical, and NMR spectroscopy. A sequential binding of Co²⁺ to the dinuclear sites in 20 mM Mes buffer at pH 6.1 has also been established. The results from these studies suggest that the two metal sites have a five-coordination sphere, with at least one coordinated His each. A di-Cu²⁺-substituted derivative of the enzyme has been prepared which exhibits a ¹H NMR spectrum with sharp hyperfine-shifted signals, again indicating the presence of a dinuclear active site. This ¹H NMR spectrum with sharp hyperfine-shifted features represents a first of its kind for a di-Cu²⁺ center in metalloproteins. Received: 19 May 1997 / Accepted: 4 September 1997     

Lipid-Membrane Affinity of Chimeric Metal-binding Green Fluorescent Protein

The Green Fluorescent Protein (GFP) is a useful marker to trace the expression of cellular proteins. However, little is known about changes in protein interaction properties after fusion to GFP. In this study, we present evidence for a binding affinity of chimeric cadmium-binding green fluorescent proteins to lipid membrane. This affinity has been observed in both cellular membranes and artificial lipid monolayers and bilayers. At the cellular level, the presence of Cd-binding peptide promoted the association of the chimeric GFP onto the lipid membrane, which declined the fluorescence emission of the engineered cells. Binding affinity to lipid membranes was further investigated using artificial lipid bilayers and monolayers. Small amounts of the chimeric GFP were found to incorporate into the lipid vesicles due to the high surface pressure of bilayer lipids. At low interfacial pressure of the lipid monolayer, incorporation of the chimeric Cd-binding GFP onto the lipid monolayer was revealed. From the measured lipid isotherms, we conclude that Cd-binding GFP mediates an increase in membrane fluidity and an expansion of the surface area of the lipid film. This evidence was strongly supported by epifluorescence microscopy, showing that the chimeric Cd-binding GFP preferentially binds to fluid-phase areas and defect parts of the lipid monolayer. All these findings demonstrate the hydrophobicity of the GFP constructs is mainly influenced by the fusion partner. Thus, the example of a metal-binding unit used here shines new light on the biophysical properties of GFP constructs.This revised version was published online in June 2005 with a corrected cover date.     

Metal-binding proteins in eggs of various sea urchin species.

Metallothionein presence and amount were determined in the unfertilized eggs of six sea urchin species by silver saturation assay and gel-chromatography of cell extracts. The results showed high levels of metallothionein in the egg cytoplasm of the two Mediterranean species Paracentrotus lividus and Sphaerechinus granularis. No metallothionein was found either in the eggs of Arbacia lixula, or in those of the three Eastern species Strongylocentrotus intermedius, Temnopleurus hardwickii and Clypeaster japonicus. However, the extracts of the latter three species revealed the presence of zinc bound in a macromolecular form, thus suggesting the existence of metal-binding proteins distinct from metallothioneins.     

Metal-binding studies for a de novo designed calcium-binding protein

To understand the key determinants in calcium-binding affinity, a calcium-binding site with pentagonal bipyramid geometry was designed into a non-calcium-binding protein, domain 1 of CD2. This metal-binding protein has five mutations with a net charge in the coordination sphere of -5 and is termed DEEEE. Fluorescence resonance energy transfer was used to determine the metal-binding affinity of DEEEE to the calcium analog terbium. The addition of protein concentration to Tb(III) solution results in a large enhancement of Tb(III) fluorescence due to energy transfer between terbium ions and aromatic residues in CD2-D1. In addition, both calcium and lanthanum compete with terbium for the same desired metal binding pocket. Our designed protein exhibits a stronger affinity for Tb(III), with a K(d) of 21 microM, than natural calcium-binding proteins with a similar Greek key scaffold.