Metal-binding Peptides Research Articles

Arsenic Species Distribution and Toxicity in the Environment, Bioaccumulation, Biomethylation and Bioremediation by Microalgae: A Review

Arsenic (As) is a noxious metalloid that has been designated a priority pollutant and is present in the environment as a consequence of both anthropogenic and natural processes. Its toxicity in environmental and biological systems depends strongly on the chemical species. Interest in arsenic and microalgae interactions is important because microalgae are at the base of the aquatic food chain, are used in animal nutrition and has potential for As phytoremediation. This paper reviewed the current information on As species distribution in the environment especially as it relates to its toxicity to microalgae as well as its bioaccumulation, biomethylation and bioremediation by microalgae using appropriate methods. Information obtained revealed that Microalgae have evolved mechanisms for dealing with As in the environment with arsBHC operon mediating the reduction and extrusion of arsenite from the cells. They accumulate large amounts of arsenic from their surroundings which could lead to toxicity, As excretion from cells, reduction, methylation or complexation with metal binding peptides like glutathione and phytochelatins. This has made them suitable as ecological indicators to give an indication of As bioavailability and also in possible applications for the process of As remediation. Microalgae are been proposed for bioremediation purposes in aquatic environment since they show a high capacity for biosorption and bioconcentration of As and most importantly since they are able to methylate inorganic As to non-toxic organic and volatile As.

Abstract 1979 Designing Metal-Binding Peptides as Dimerization Disruptors of Ribose-5-Phosphate Isomerase B from Leishmania donovani

Abstract 1979 Designing Metal-Binding Peptides as Dimerization Disruptors of Ribose-5-Phosphate Isomerase B from Leishmania donovani

Synthesis of metal-binding amino acids.

The ability for amino acid residues to bind metals underpins the functions of metalloproteins to conduct a plethora of critical processes in living organisms as well as unnatural applications in the fields of catalysis, sensing and medicinal chemistry. The capability to access metal-binding peptides heavily relies on the ability to generate appropriate building blocks. This review outlines recently developed strategies for the synthesis of metal binding non-proteinogenic amino acids. The chemistries to access, as well as to incorporate these amino acids into peptides is presented herein.

Gas-phase Ni(II) affinities of alternative metal binding peptides from competitive threshold collision-induced dissociation

The Ni(II) affinity of the polyhistidine tag is used in the purification of recombinant proteins by immobilized metal affinity chromatography. Here we measured the relative gas-phase Ni(II) affinities of four alternative metal binding (amb) peptides and the 7xHis tag using the competitive threshold collision-induced dissociation (TCID) technique. The general primary structure of the four amb peptides was acetyl-Aa1-Aa2-Gly3-Pro4-Aa5-Gly6-Cys7, designed to test whether the His1-Cys2 or Asp1-His2 would exhibit the higher Ni(II) affinity and whether the Tyr5 with its ability of forming long-range π-nickel interaction and hydrogen bonding would contribute to the Ni(II) affinity. The Cys7 is retained in all the amb sequences because previous research has shown that both the thiolate side group and carboxylate terminus simultaneously coordinate the metal ion. The Ni(II) affinity was measured using the dissociation of the [amb + Ni(II) + NTA]− complex, where NTA = nitrilotriacetic acid, which is a commonly used ligand for Ni(II) inside the IMAC column. The dissociation of [amb + Ni(II) + NTA]− produced two main product channels; [amb + Ni(II)]− + NTA and [NTA + Ni(II)]− + amb, whose competition was modeled by TCID to extract the relative gas-phase Ni(II) affinities. Extensive molecular modeling using PM6 located the low-energy structures whose molecular parameters were used in the TCID analyses if their collision cross sections agreed with those measured by traveling-wave ion mobility mass spectrometry and they were compatible with a concerted reaction. We compare the final results by conducting the TCID analyses using alternative PM6 parameters and by making the ternary complexes in acidic and basic solutions.

Metal-binding peptides and their potential to enhance the absorption and bioavailability of minerals.

Minerals including calcium, iron, zinc, magnesium, and copper have several human nutritional functions due to their metabolic activities. Body tissues require sufficient levels of a variety of micronutrients to maintain their health. To achieve these micronutrient needs, dietary consumption must be adequate. Dietary proteins may regulate the biological functions of the body in addition to acting as nutrients. Some peptides encoded in the native protein sequences are primarily responsible for the absorption and bioavailability of minerals in physiological functions. Metal-binding peptides (MBPs) were discovered as potential agents for mineral supplements. Nevertheless, sufficient studies on how MBPs affect the biological functions of minerals are lacking. The hypothesis is that the absorption and bioavailability of minerals are significantly influenced by peptides, and these properties are further enhanced by the configuration and attribute of the metal-peptide complex. In this review, the production of MBPs is discussed using various key parameters such as the protein sources and amino acid residues, enzymatic hydrolysis, purification, sequencing and synthesis and in silico analysis of MBPs. The mechanisms of metal-peptide complexes as functional food ingredients are elucidated, including metal-peptide ratio, precursors and ligands, complexation reaction, absorbability and bioavailability. Finally, the characteristics and application of different metal-peptide complexes are also described.

Flow cytometry-based high-throughput screening of synthetic peptides for palladium adsorption

Conventionally, the measurement of metal ion adsorption capacity in biosorbent relies on expensive and time-consuming ICP-OES technique. Herein, a semi-quantitative method to measure Pd(II) adsorption capacity of single cells has been presented by analyzing side scatter (SSC) intensity in flow cytometry. Within the sensitive range and applicable conditions, excellent linearity correlation (R2 ranges from 0.89 to 0.96) between the amount of Pd(II) absorbed on yeast and the fold increase in SSC intensity has been observed. Using this method, six strains with high Pd adsorption capacities have sorted from a yeast library with metal-binding peptides displayed (up to 107 strains) based on SSC signal intensity. The optimal peptide (EF1) displayed on yeast and E. coli surface demonstrated Pd adsorption improvements of ∼32% and ∼200%, respectively. In summary, our study proposes an alternative high-throughput method for analyzing the Pd(II) adsorption capacity of individual yeast cells, enabling the screening of specific peptides/proteins with high Pd(II) affinity from extensive libraries.

Discovery of phytochelatins in human urine: Evidence for function in selenium disposition and protection against cadmium.

This report identifies, for the first time, a phytochelatin compound, phytochelatin 2 [γ-E-C-γ-E-C-G], and related metabolites in human urine. Phytochelatins are metal-binding peptides produced by plants. They are present in nearly all human diets, due to their ubiquity in plants. The urinary concentration of phytochelatin 2 among 143 adults was in the low micromolar range, and phytochelatin 2 and its metabolites had differential correlations with urinary selenium and toxic metals. Activities of ingested phytochelatins are largely undescribed. Observed urinary metal interactions were investigated further in cell and animal models. Selenite reacted with phytochelatin to form a phytochelatin selenotrisulfide, and the preformed selenotrisulfide showed increased selenium uptake by renal proximal tubule cells. In vivo studies further showed that oral phytochelatin increased renal selenium content and decreased lung cadmium in mice. Presence of phytochelatin in human urine combined with its function in selenium and heavy metal distribution present a new route by which diet may influence metal disposition and bioavailability.

Improving dental epithelial junction on dental implants with bioengineered peptides.

Introduction: The functionalization of titanium (Ti) and titanium alloys (Ti6Al4V) implant surfaces via material-specific peptides influence host/biomaterial interaction. The impact of using peptides as molecular linkers between cells and implant material to improve keratinocyte adhesion is reported. Results: The metal binding peptides (MBP-1, MBP-2) SVSVGMKPSPRP and WDPPTLKRPVSP were selected via phage display and combined with laminin-5 or E-cadherin epithelial cell specific peptides (CSP-1, CSP-2) to engineer four metal-cell specific peptides (MCSPs). Single-cell force spectroscopy and cell adhesion experiments were performed to select the most promising candidate. In vivo tests using the dental implant for rats showed that the selected bi functional peptide not only enabled stable cell adhesion on the trans-gingival part of the dental implant but also arrested the unwanted apical migration of epithelial cells. Conclusion: The results demonstrated the outstanding performance of the bioengineered peptide in improving epithelial adhesion to Ti based implants and pointed towards promising new opportunities for applications in clinical practice.

Entropy-enthalpy interplay in aluminum bound neurofilaments.

Considerable amount of work has been shown to understand the preferred chelation modes of metal-binding peptides using quantum mechanical calculations, resulting in a hierarchy of binding affinities. Previous work has shown an increasing stabilization energy for higher coordination complexes. However, as the coordination of a metal increases, the conformational space available for the polypeptide chain is inevitably reduced. Resolving the many entropic contributions from different degrees of freedom is particularly challenging. In this work, we explore this problem focusing on a family of phosphorylated neuropeptides that bind to aluminum. Determination of the configurational entropy is done using Maximum Information Spanning Tree (MIST) scheme implemented in the parallel software PARENT. We find that there is a general negative correlation between both stabilization energy and entropy, and that a subtle interplay determines the population of the most favorable species, rewarding a balance between both contributions. Additionally, we synthesize and discuss the requirements for a possible Metal Ion Hypothesis based on our findings.

Concentrations of Transition Metal Ions in Rat Lungs after Tobacco Smoke Exposure and Treatment with His-Leu Dipeptide.

Tobacco smoking is deleterious to the lungs because it exposes them to many toxic substances. These include transition metal ions, such as cadmium. However, there is a lack of information about the influence of endogenous metal-binding peptides, such as His-Leu (HL), on the lung distribution of transition metals in smokers. To address this, we administered HL subcutaneously to rats exposed to tobacco smoke for six weeks, then we measured the concentrations of transition metal ions in the lungs. We found that exposure to tobacco smoke elevates the concentrations of Cd(II) and Cu(II). Administration of the HL peptide, whose elevation is a consequence of angiotensin receptor blocker anti-hypertension therapy, increases the concentration of Fe in the lungs of rats exposed to smoke. These findings suggest that smoking is a risk factor for patients receiving angiotensin receptor blockers to treat hypertension.

Influence of metal binding on the conformational landscape of neurofilament peptides.

In order to understand the preferred modes of chelation in metal-binding peptides, quantum mechanical calculations can be used to compute energies, resulting in a hierarchy of binding affinities. These calculations often produce increasing stabilization energies the higher the coordination of the complex. However, as the coordination of a metal increases, the conformational freedom of the polypeptide chain is inevitably reduced, resulting in an entropic penalty. Estimating the magnitude of this penalty from the many different degrees of freedom of biomolecular systems is very challenging, and as a result this contribution to the free energy is often ignored. Here we explore this problem focusing on a family of phosphorylated neuropeptides that bind to aluminum. We find that there is a general negative correlation between both stabilization energy and entropy. Our results suggest that a subtle interplay between enthalpic and entropic forces will determine the population of the most favourable species. Additionally, we discuss the requirements for a possible "Metal Ion Hypothesis" based on our findings.

Sequence rules for gold-binding peptides.

Metal-binding peptides play a central role in bionanotechnology, wherein they are responsible for directing growth and influencing the resulting properties of inorganic nanomaterials. One of the key advantages of using peptides to create nanomaterials is their versatility, wherein subtle changes in the sequence can have a dramatic effect on the structure and properties of the nanomaterial. However, precisely knowing which position and which amino acid should be modified within a given sequence to enhance a specific property can be a daunting challenge owing to combinatorial complexity. In this study, classification based on association rules was performed using 860 gold-binding peptides. Using a minimum support threshold of 0.035 and confidence of 0.9, 30 rules with confidence and lift values greater than 0.9 and 1, respectively, were extracted that can differentiate high-binding from low-binding peptides. The test performance of these rules for categorizing the peptides was found to be satisfactory, as characterized by accuracy = 0.942, F1 = 0.941, MCC = 0.884. What stands out from the extracted rules are the importance of tryptophan and arginine residues in differentiating peptides with high binding affinity from those with low affinity. In addition, the association rules revealed that positions 2 and 4 within a decapeptide are frequently involved in the rules, thus suggesting their importance in influencing peptide binding affinity to AuNPs. Collectively, this study identified sequence rules that may be used to design peptides with high binding affinity.

The Use of a Replicating Virus Vector For in Planta Generation of Tobacco Mosaic Virus Nanorods Suitable For Metallization.

The production of designer-length tobacco mosaic virus (TMV) nanorods in plants has been problematic in terms of yields, particularly when modified coat protein subunits are incorporated. To address this, we have investigated the use of a replicating potato virus X-based vector (pEff) to express defined length nanorods containing either wild-type or modified versions of the TMV coat protein. This system has previously been shown to be an efficient method for producing virus-like particles of filamentous plant viruses. The length of the resulting TMV nanorods can be controlled by varying the length of the encapsidated RNA. Nanorod lengths were analyzed with a custom-written Python computer script coupled with the Nanorod UI user interface script, thereby generating histograms of particle length. In addition, nanorod variants were produced by incorporating coat protein subunits presenting metal-binding peptides at their C-termini. We demonstrate the utility of this approach by generating nanorods that bind colloidal gold nanoparticles.

Systemic Regulation of Iron Acquisition by Arabidopsis in Environments with Heterogeneous Iron Distributions.

Nutrient distribution within the soil is generally heterogeneous. Plants, therefore, have evolved sophisticated systemic processes enabling them to optimize their nutrient acquisition efficiency. By organ-to-organ communication in Arabidopsis thaliana, for instance, iron (Fe) starvation in one part of a root drives the upregulation of a high-affinity Fe-uptake system in other root regions surrounded by sufficient levels of Fe. This compensatory response through Fe-starvation-triggered organ-to-organ communication includes the upregulation of Iron-regulated transporter 1 (IRT1) gene expression on the Fe-sufficient side of the root; however, the molecular basis underlying this long-distance signaling remains unclear. Here, we analyzed gene expression by RNA-seq analysis of Fe-starved split-root cultures. Genome-wide expression analysis showed that localized Fe depletion in roots upregulated several genes involved in Fe uptake and signaling, such as IRT1, in a distant part of the root exposed to Fe-sufficient conditions. This result indicates that long-distance signaling for Fe demand alters the expression of a subset of genes responsible for Fe uptake and coumarin biosynthesis to maintain a level of Fe acquisition sufficient for the entire plant. Loss of IRON MAN/FE-UPTAKE-INDUCING PEPTIDE (IMA/FEP) leads to the disruption of compensatory upregulation of IRT1 in the root surrounded by sufficient Fe. In addition, our split-root culture-based analysis provides evidence that the IMA3/FEP1-MYB10/72 pathway mediates long-distance signaling in Fe homeostasis through the regulation of coumarin biosynthesis. These data suggest that the signaling of IMA/FEP, a ubiquitous family of metal-binding peptides, is critical for organ-to-organ communication in response to Fe starvation under heterogeneous Fe conditions in the surrounding environment.

Thermochemical and conformational studies of Ni(II) and Zn(II) ternary complexes of alternative metal binding peptides with nitrilotriacetic acid

Energy-resolved threshold collision-induced dissociation (TCID) of negatively-charged [amb5-H+M(II)+NTA-2H]− ternary complexes were studied for how they competitively dissociated into the products [amb5-3H+M(II)]− + NTA or [NTA-3H+M(II)]− + amb5, where M = Zn or Ni and NTA is nitrilotriacetic acid. The complexes contained one of the alternative metal binding-5 (amb5) heptapeptides with primary structures acetyl-Aa1-Aa2-Gly3-Pro4-Tyr5-Aa6-Aa7, where the amino acids Aa1,2,6,7 included the potential metal binding sites of Asp, His, and/or Cys. The study included heptahistidine which represents the His affinity tag commonly used in immobilized metal affinity chromatography (IMAC) separation of recombinant proteins. Molecular modeling using PM6 located low energy, geometry-optimized conformers of the ternary complexes and their products which had collision cross sections that agreed with those measured by ion mobility. Using the molecular parameters, including vibrational and rotational frequencies, of the reactant and products from the molecular modeling, the energy-dependent intensities of the two product channels were modeled using a competitive TCID method to determine their threshold energies which equated to the two product channels 0 K dissociation enthalpies (ΔH0). Statistical mechanics thermal corrections provided the 298 K enthalpies (ΔH298) and Gibbs free energies (ΔG298) for the association reactions [amb5-3H+M(II)]− + NTA ↔ [amb5-H+M(II)+NTA-2H]− and [NTA-3H+M(II)]− + amb5 ↔ [amb5-H+M(II)+NTA-2H]−. These results were compared to determine which amb5 were effective as alternative tags for IMAC purification of recombinant proteins and is a routine that can be generally applied to compare the thermochemistry of metal ion ternary complexes.

Copper-Binding Peptides Attenuate Microglia Inflammation through Suppression of NF-kB Pathway.

Activation of microglia, the resident immune cells of the central nervous system, has been related to the etiology and progression of neurodegenerative diseases; thus, finding novel approaches to suppress the neuroinflammatory process is of utmost relevance. The anti-inflammatory activity of whey Cu-, Fe-, and Zn-binding peptides and their possible underlying mechanism of action were evaluated in microglia. Whey metal-binding peptides decreased nitric oxide production and tumor necrosis factor α (TNF-α) at mRNA and protein levels by stimulated BV-2 microglia in comparison to the control with no peptide treatment. The hydrophobicity, specific sequences, and possible synergistic effects seem to play a role. Cu-binding peptides (Cu-bp) presented anti-inflammatory activity both in BV-2 and primary microglia cultures. These peptides exert their action by suppressing nuclear factor kappa B (NF-kB) pathway since nuclear translocation of NF-kB p65 is decreased by roughly 30% upon Cu-bp treatment. Specific sequences identified in Cu-bp showed high affinity to bind NF-kB p65 by molecular docking (up to -8.8kcal mol-1 ), corroborating the immunofluorescence studies. Cu-bp represent food-derived peptides that may be useful for neuroprotective purposes. Chelation of copper excess in the CNS and the bioavailability of such peptides, as well as their behavior in in vivo models, deserve further research for future applications.

Analyzing cadmium-phytochelatin2 complexes in plant using terahertz and circular dichroism information

Phytochelatins are plants’ small metal-binding peptides which chelate internal heavy metals to form nontoxic complexes. Detecting the complexes in plants would simplify identification of cultivars with both high tolerance and enrichment capabilities for heavy metals which represent phytoextraction performance. Thus, a terahertz spectroscopy combined with density functional theory, chemometrics and circular dichroism was used for characterization of phytochelatin2 (PC2), Cd-PC2 mixture standards, and pak choi (Brassica chinensis) leaves as a plant model. Results showed PC2 chelates Cd2+ in a 2:1 ratio to form Cd(PC2)2 complex; Cd connected to thoils of PC2 and changed β-turn and random coil of PC2 peptide chain to β-Sheet which presented as terahertz vibrations of PC2 around 1.03 and 1.71 THz being suppressed; the best models for detecting the complex in pak choi were obtained by partial least squares regression modeling combined with successive projections algorithm selection; the models used PC2 as a natural probe for visualizing and quantifying chelated Cd in pak choi leaf and achieved a limit of detection up to 1.151 ppm. This study suggested that terahertz information of the heavy metal-PCs complexes is qualified for representing a simpler alternative to classical index for evaluating phytoextraction performance of plant; it provided a general protocol for structure analysis and detection of heavy metal-PCs complexes in plant by terahertz absorbance.

Expression regulation of chitin-binding protein and metal-binding peptide in new Bacillus velezensis: MALDI-TOF MS/MS analysis.

Metallothionein and metal-binding peptides are small cysteine-rich proteins produced by different organisms in stress conditions. In this study, the metal-binding peptide was detected in extracellular proteins of a new Bacillus velezensis strain, isolated from metal contaminated soil, and grown on the lead-enriched medium, for the first time. The presence of sulfide peptide was assayed by two simple tests (lead sulfide and Ellman's reagent test) for preliminary, and subsequently confirmed using polyacrylamide gel electrophoresis at media with different lead concentrations that the low-molecular-weight protein fragments (≈10 kDa) were observed while none were detected in the medium containing sodium chloride or calcium salt. The amino acids of the observed fragments were analyzed by matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS). Also, the metal adsorption was confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by staining with chromium solution. The results showed that the putative sulfide peptide is metallothionein, which is induced in stress conditions. It was interesting that in all SDS profiles, one protein fragment (≈18 kDa) was inhibited in lead-enriched media. The data from MALDI-TOF MS/MS analysis showed that this fraction was a chitin-binding protein whose production was regulated by metal contamination. It is anticipated that these two proteins regulate the toxicity of lead.

Heavy metal stress in rice: Uptake, transport, signaling, and tolerance mechanisms.

Heavy metal contamination of agricultural fields has become a global concern as it causes a direct impact on human health. Rice is the major food crop for almost half of the world population and is grown under diverse environmental conditions, including heavy metal-contaminated soil. In recent years, the impact of heavy metal contamination on rice yield and grain quality has been shown through multiple approaches. In this review article, different aspects of heavy metal stress, that is uptake, transport, signaling and tolerance mechanisms, are comprehensively discussed with special emphasis on rice. For uptake, some of the transporters have specificity to one or two metal ions, whereas many other transporters are able to transport many different ions. After uptake, the intercellular signaling is mediated through different signaling pathways involving the regulation of various hormones, alteration of calcium levels, and the activation of mitogen-activated protein kinases. Heavy metal stress signals from various intermediate molecules activate various transcription factors, which triggers the expression of various antioxidant enzymes. Activated antioxidant enzymes then scavenge various reactive oxygen species, which eventually leads to stress tolerance in plants. Non-enzymatic antioxidants, such as ascorbate, metalloids, and even metal-binding peptides (metallothionein and phytochelatin) can also help to reduce metal toxicity in plants. Genetic engineering has been successfully used in rice and many other crops to increase metal tolerance and reduce heavy metals accumulation. A comprehensive understanding of uptake, transport, signaling, and tolerance mechanisms will help to grow rice plants in agricultural fields with less heavy metal accumulation in grains.

Cloning of PCS gene (TpPCS1) from Tagetes patula L. and expression analysis under cadmium stress.

Phytochelatins (PCs) constitute an important mechanism for plants to resist heavy metal stress. Widely found in higher plants, they are small heavy metal binding peptides, synthesized through catalysis of phytochelatin synthase (PCS). We speculate that there may be PCS genes in Peacock grass (Tagetes patula L., Asteraceae), which is an important reason for its rich cadmium. In order to obtain the full-length cDNA sequence of the PCS gene from T. patula L. used rapid amplification of cDNA ends (RACE). Meanwhile, Relative expression of TpPCS1 under different concentrations of cadmium (Cd) stress was analysed using quantitative real-time polymerase chain reaction (qRT-PCR). Results found ORF of TpPCS1 genes with a length of 1970 bp, a gene coding area length of 1764 bp, coding for 587 amino acids. Expression of TpPCS1 under Cd stress was tissue specific. TpPCS1 in the root showed higher expression, while expression in the leaf and seed was relatively low. This research demonstrates that expression of TpPCS1 enhanced the enrichment of cadmium in T. patula L. roots and could be used to construct a plant hyperexpression carrier that would provide new avenues for plant restoration technology.