Microbial green synthesis of luminescent terbium sulfide nanoparticles using E. Coli: a rare earth element detoxification mechanism.

BACKGROUND: Rare-earth sulfide nanoparticles (NPs) could harness the optical and magnetic features of rare-earth ions for applications in nanotechnology. However, reports of their synthesis are scarce and typically require high temperatures and long synthesis times. RESULTS: Here we present a biosynthesis of terbium sulfide (TbS) NPs using microorganisms, identifying conditions that allow Escherichia coli to extracellularly produce TbS NPs in aqueous media at 37 °C by controlling cellular sulfur metabolism to produce a high concentration of sulfide ions. Electron microscopy revealed ultrasmall spherical NPs with a mean diameter of 4.1 ± 1.3 nm. Electron diffraction indicated a high degree of crystallinity, while elemental mapping confirmed colocalization of terbium and sulfur. The NPs exhibit characteristic absorbance and luminescence of terbium, with downshifting quantum yield (QY) reaching 28.3% and an emission lifetime of ~ 2 ms. CONCLUSIONS: This high QY and long emission lifetime is unusual in a neat rare-earth compound; it is typically associated with rare-earth ions doped into another crystalline lattice to avoid non-radiative cross relaxation. This suggests a reduced role of nonradiative processes in these terbium-based NPs. This is, to our knowledge, the first report revealing the advantage of biosynthesis over chemical synthesis for Rare Earth Element (REE) based NPs, opening routes to new REE-based nanocrystals.

Enrichment of Terbium(III) under synergistic effect of biosorption and biomineralization by Bacillus sp. DW015 and Sporosarcina pasteurii.

Biosorption and biomineralization are commonly used for the immobilization of metal ions. Biosorption is commonly used as a green method to enrich rare earth ions from wastewater. However, little attention has been paid to the facilitating role of biomineralization in the enrichment of rare earth ions. In this study, a strain of Bacillus sp. DW015, isolated from ion adsorption type rare earth ores and a urease-producing strain Sporosarcina pasteurii were used to enrich rare earth elements (REEs) from an aqueous solution. The results indicate that biomineralization accelerates the enrichment of Terbium(III) compared to biosorption alone. Kinetic analysis suggests that the main mode of action of DW015 was biosorption, following pseudo-second-order kinetics (R2 = 0.998). The biomineralization of DW015 did not significantly contribute to the enrichment of Tb(III), whereas excessive biomineralization of S. pasteurii led to a decrease in the enrichment of Tb(III). A synergistic system of biosorption and biomineralization was established by combining the two bacteria, with the optimal mixed bacteria (S. pasteurii:DW015) ratio being 1:19. This study provides fundamental support for the synergistic effect of biosorption and biomineralization and offers a new reference for future microbial-based enrichment methods. IMPORTANCE: A weak microbially induced calcium carbonate precipitation (MICP) promotes the enrichment of Tb(III) by bacteria, while a strong MICP leads to the release of Tb(III). However, existing explanations cannot elucidate these mechanisms. In this study, the morphology of the bioprecipitation and the degree of Tb(III) enrichment were analyzed by X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS). The data revealed that MICP could drive stable attachment of Tb(III) onto the cell surface, forming a Tb-CaCO3 mixed solid phase. Excessive rapid rate of calcite generation could disrupt the Tb(III) adsorption equilibrium, leading to the release of Tb(III). Therefore, in order for Tb(III) to be stably embedded in calcite, it is necessary to have a sufficient number of adsorption sites on the bacteria and to regulate the rate of MICP. This study provides theoretical support for the process design of MICP for the enrichment of rare earth ions.

How predicted temperature and salinity changes will modulate the impacts induced by terbium in bivalves?

The threat of climate change, which includes shifts in salinity and temperature, has generated a global concern for marine organisms. These changes directly impact them and may alter their susceptibility to contaminants, such as terbium (Tb), found in electronic waste. This study assessed how decreased and increased salinity, as well as increased temperature, modulates Tb effects in Mytilus galloprovincialis mussels. After an exposure period of 28 days, Tb bioaccumulation and biochemical changes were evaluated. Results indicated no significant modulation of salinity and temperature on Tb accumulation, suggesting detoxification mechanisms and adaptations. Further analysis showed that Tb exposure alone caused antioxidant inhibition and neurotoxicity. When exposed to decreased salinity, these Tb-exposed organisms activated defense mechanisms, a response indicative of osmotic stress. Moreover, increased salinity also led to increased oxidative stress and metabolic activity in Tb-exposed organisms. Additionally, Tb-exposed organisms responded to elevated temperature with altered biochemical activities indicative of damage and stress response. Such responses suggested that Tb effects were masked by osmotic and heat stress. This study provides valuable insights into the interactions between temperature, salinity, and contaminants such as Tb, impacting marine organisms. Understanding these relationships is crucial for mitigating climate change and electronic waste effects on marine ecosystems.

Insight into effects of terbium on cell growth, sporulation and spore properties of Bacillus subtilis.

Recovery of rare earth elements (REEs) from wastewater with Bacillus subtilis (B. subtilis) during culture is promising due to its environmental benefits. However, the effects of REEs in the culture media on B. subtilis are poorly understood. This study aims to investigate the effects of the terbium (Tb(III)), a typical rare earth element, on the cell growth, sporulation, and spore properties of B. subtilis. Tb(III) can suppress bacterial growth while enhancing spore tolerance to wet heat. Spore germination and content of dipicolinic acid (DPA) were promoted at low concentrations of Tb(III) while inhibited at a high level, but an inverse effect on initial sporulation appeared. Scanning electron microscope and energy dispersive spectrometer detection indicated that Tb(III) complexed cells or spores and certain media components simultaneously. The germination results of the spores after elution revealed that Tb(III) attached to the spore surface was a key effector of spore germination. In conclusion, Tb(III) directly or indirectly regulated both the nutrient status of the media and certain metabolic events, which in turn affected most of the properties of B. subtilis. Compared to the coat-deficient strain, the wild-type strain grew faster and was more tolerant to Tb(III), DPA, and wet heat, which in turn implied that it was more suitable for the recovery of REEs during cultivation. These findings provide fundamental insights for the recovery of rare earths during the culture process using microorganisms.

Therapeutic potentials of terbium hydroxide nanorods for amelioration of hypoxia-reperfusion injury in cardiomyocytes.

Myocardial hypoxia reperfusion (H/R) injury is the paradoxical exacerbation of myocardial damage, caused by the sudden restoration of blood flow to hypoxia affected myocardium. It is a critical contributor of acute myocardial infarction, which can lead to cardiac failure. Despite the current pharmacological advancements, clinical translation of cardioprotective therapies have proven challenging. As a result, researchers are looking for alternative approaches to counter the disease. In this regard, nanotechnology, with its versatile applications in biology and medicine, can confer broad prospects for treatment of myocardial H/R injury. Herein, we attempted to explore whether a well-established pro-angiogenic nanoparticle, terbium hydroxide nanorods (THNR) can ameliorate myocardial H/R injury. For this study, in vitro H/R-injury model was established in rat cardiomyocytes (H9c2 cells). Our investigations demonstrated that THNR enhance cardiomyocyte survival against H/R-induced cell death. This pro-survival effect of THNR is associated with reduction of oxidative stress, lipid peroxidation, calcium overload, restoration of cytoskeletal integrity and mitochondrial membrane potential as well as augmentation of cellular anti-oxidant enzymes such as glutathione-s-transferase (GST) and superoxide dismutase (SOD) to counter H/R injury. Molecular analysis revealed that the above observations are traceable to the predominant activation of PI3K-AKT-mTOR and ERK-MEK signalling pathways by THNR. Concurrently, THNR also exhibit apoptosis inhibitory effects mainly by suppression of pro-apoptotic proteins like Cytochrome C, Caspase 3, Bax and p53 with simultaneous restoration of anti-apoptotic protein, Bcl-2 and Survivin. Thus, considering the above attributes, we firmly believe that THNR have the potential to be developed as an alternative approach for amelioration of H/R injury in cardiomyocytes.

Assessing the impact of terbium on Mytilus galloprovincialis: Metabolic and oxidative stress responses.

The increasing demand for electric and electronic equipment has led to a rise in potentially hazardous electronic waste, including rare-earth elements (REEs), such as terbium (Tb), which have been already detected in aquatic systems. This study investigated the biochemical effects of anthropogenic Tb on mussels over a 28-day period. The mussels were exposed to different concentrations of Tb (0, 5, 10, 20, 40 µg/L), and biomarkers related to metabolism, oxidative stress, cellular damage, and neurotoxicity were evaluated. Bioaccumulation of Tb in the mussels' tissue increased with exposure concentrations, but the bioconcentration factor remained similar between treatments. Exposure to Tb enhanced glycogen consumption and decreased metabolic capacity which could be seen as a physiological adaptation to limit Tb accumulation. Antioxidant defenses and glutathione S-transferases showed a more complex dose-response, with enzymatic responses increasing until 10 µg/L but then returning to control levels at 20 µg/L. At 40 µg/L, enzymatic responses were also enhanced but to a lower extent than at 10 µg/L. The presence of Tb had clearly an inhibitory effect on biotransformation enzymes such as carboxylesterases in a dose-dependent manner. Likely, thanks to biochemical and physiological adaptations, no cellular damage or neurotoxicity was observed in any treatments, confirming the mussels' ability to tolerate Tb exposure. Nevertheless, prolonged exposure to these concentrations could lead to harmful consequences when facing other environmental stressors, such as misallocating energy resources for growth, reproduction, and defense mechanisms.

Systematic detection of tertiary structural modules in large RNAs and RNP interfaces by Tb-seq.

Compact RNA structural motifs control many aspects of gene expression, but we lack methods for finding these structures in the vast expanse of multi-kilobase RNAs. To adopt specific 3-D shapes, many RNA modules must compress their RNA backbones together, bringing negatively charged phosphates into close proximity. This is often accomplished by recruiting multivalent cations (usually Mg2+), which stabilize these sites and neutralize regions of local negative charge. Coordinated lanthanide ions, such as terbium (III) (Tb3+), can also be recruited to these sites, where they induce efficient RNA cleavage, thereby revealing compact RNA 3-D modules. Until now, Tb3+ cleavage sites were monitored via low-throughput biochemical methods only applicable to small RNAs. Here we present Tb-seq, a high-throughput sequencing method for detecting compact tertiary structures in large RNAs. Tb-seq detects sharp backbone turns found in RNA tertiary structures and RNP interfaces, providing a way to scan transcriptomes for stable structural modules and potential riboregulatory motifs.

Multi-Functional Silver Nanoparticles for High-Throughput Endospore Sensing.

In spore-forming bacteria such as Bacillus and Clostridium, the vegetative cells form highly durable hard shells called endospores inside the bacteria to survive as the growth environment deteriorates. Because of these properties, endospores can cause food poisoning and medical accidents if they contaminate food, medicine, or other products, and it is required for technology to detect the spores at the manufacturing site. In this study, we focused on the surface-enhanced Raman scattering (SERS) method for the sensitive detection of dipicolinic acid (DPA), a molecular marker of endospores. We constructed Fe3O4/Ag core-shell functional silver nanoparticles that specifically bind to DPA, and investigated a method for the qualitative detection of DPA by SERS and the quantitative detection of DPA by fluorescence method using a terbium complex formed on the surface. As a result, the concentration of the functional silver nanoparticles constructed could detect spore-derived DPA by fluorescence detection method, and SERS was several tens of nM. The functionalized nanoparticles can detect DPA quantitatively and qualitatively, and are expected to be applied to detection technology in the production of food and pharmaceuticals.

Probing Lanmodulin's Lanthanide Recognition via Sensitized Luminescence Yields a Platform for Quantification of Terbium in Acid Mine Drainage.

Lanmodulin is the first natural, selective macrochelator for f elements-a protein that binds lanthanides with picomolar affinity at 3 EF hands, motifs that instead bind calcium in most other proteins. Here, we use sensitized terbium luminescence to probe the mechanism of lanthanide recognition by this protein as well as to develop a terbium-specific biosensor that can be applied directly in environmental samples. By incorporating tryptophan residues into specific EF hands, we infer the order of metal binding of these three sites. Despite lanmodulin's remarkable lanthanide binding properties, its coordination of approximately two solvent molecules per site (by luminescence lifetime) and metal dissociation kinetics (koff = 0.02-0.05 s-1, by stopped-flow fluorescence) are revealed to be rather ordinary among EF hands; what sets lanmodulin apart is that metal association is nearly diffusion limited (kon ≈ 109 M-1 s-1). Finally, we show that Trp-substituted lanmodulin can quantify 3 ppb (18 nM) terbium directly in acid mine drainage at pH 3.2 in the presence of a 100-fold excess of other rare earths and a 100 000-fold excess of other metals using a plate reader. These studies not only yield insight into lanmodulin's mechanism of lanthanide recognition and the structures of its metal binding sites but also show that this protein's unique combination of affinity and selectivity outperforms synthetic luminescence-based sensors, opening the door to rapid and inexpensive methods for selective sensing of individual lanthanides in the environment and in-line monitoring in industrial operations.

Effective binding of Tb3+ and La3+ cations on the donor side of Mn-depleted photosystem II.

The interaction of Tb3+ and La3+ cations with different photosystem II (PSII) membranes (intact PSII, Ca-depleted PSII (PSII[-Ca]) and Mn-depleted PSII (PSII[-Mn]) membranes) was studied. Although both lanthanide cations (Ln3+) interact only with Ca2+-binding site of oxygen-evolving complex (OEC) in PSII and PSII(-Ca) membranes, we found that in PSII(-Mn) membranes both Ln3+ ions tightly bind to another site localized on the oxidizing side of PSII. Binding of Ln3+ cations to this site is not protected by Ca2+ and is accompanied by very effective inhibition of Mn2+ oxidation at the high-affinity (HA) Mn-binding site ([Mn2+ + H2O2] couple was used as a donor of electrons). The values of the constant for inhibition of electron transport Ki are equal to 2.10 ± 0.03 µM for Tb3+ and 8.3 ± 0.4 µM for La3+, whereas OEC inhibition constant in the native PSII membranes is 323 ± 7 µM for Tb3+. The value of Ki for Tb3+ corresponds to Ki for Mn2+ cations in the reaction of diphenylcarbazide oxidation via HA site (1.5 µM) presented in the literature. Our results suggest that Ln3+ cations bind to the HA Mn-binding site in PSII(-Mn) membranes like Mn2+ or Fe2+ cations. Taking into account the fact that Mn2+ and Fe2+ cations bind to the HA site as trivalent cations after light-induced oxidation and the fact that Mn cation bound to the HA site (Mn4) is also in trivalent state, we can suggest that valency may be important for the interaction of Ln3+ with the HA site.

In Vivo Shaping of Inorganic Functional Devices using Microalgae.

The usage of biomineralization processes performed by living microalgae to create 3D nanostructured materials are advantageous compared to conventional synthesis routes. Exploitation of in vivo shaping using living cells leads to inorganic intricate biominerals, produced with low environmental impact. Since biomineralization processes are genetically controlled, the formation of nanostructured materials is highly reproducible. The shells of microalgae, like coccoliths, are particularly of great interest. This study shows the generation of mesoporous highly structured functional materials with induced optoelectronical properties using in vivo processes of the microalga species Emiliania huxleyi. It demonstrates the metabolically driven incorporation of the lanthanide terbium into the coccoliths of E. huxleyi as a route for the synthesis of finely patterned photoluminescent particles by feeding the microalgae with this luminescent element. The resulting green luminescent particles have hierarchical ordered pores on the nano- and microscale and may act as powerful tools for many applications; they may serve as imaging probes for biomedical applications, or in microoptics. The luminescent coccoliths combine a unique hierarchical structure with a characteristic luminescence pattern, which make them superior to conventional produced Tb doted material. With this study, the possibility of the further exploitation of coccoliths as advanced functional materials for nanotechnological applications is given.

Design of a Protein Motif Responsive to Tyrosine Nitration and an Encoded Turn-Off Sensor of Tyrosine Nitration.

Tyrosine nitration is a protein post-translational modification that is predominantly non-enzymatic and is observed to be increased under conditions of nitrosative stress and in numerous disease states. A small protein motif (14-18 amino acids) responsive to tyrosine nitration has been developed. In this design, nitrotyrosine replaced the conserved Glu12 of an EF-hand metal-binding motif. Thus, the non-nitrated peptide bound terbium weakly. In contrast, tyrosine nitration resulted in a 45-fold increase in terbium affinity. Nuclear magnetic resonance spectroscopy indicated direct binding of nitrotyrosine to the metal and EF-hand-like metal contacts in this designed peptide. Nitrotyrosine is an efficient quencher of fluorescence. To develop a sensor of tyrosine nitration, the initial design was modified to incorporate Glu residues at EF-hand positions 9 and 16 as additional metal-binding residues, to increase the terbium affinity of the peptide with unmodified tyrosine. This peptide with a tyrosine at residue 12 bound terbium and effectively sensitized terbium luminescence. Tyrosine nitration resulted in a 180-fold increase in terbium affinity ( Kd = 1.6 µM) and quenching of terbium luminescence. This sequence was incorporated as an encoded protein tag and applied as a turn-off fluorescent protein sensor of tyrosine nitration. The sensor was responsive to nitration by peroxynitrite, with fluorescence quenched upon nitration. The greater terbium affinity upon tyrosine nitration resulted in a large dynamic range and sensitivity to substoichiometric nitration. An improved approach for the synthesis of peptides containing nitrotyrosine was also developed, via the in situ silyl protection of nitrotyrosine. This work represents the first designed, encodable protein motif that is responsive to tyrosine nitration.

Non-Additive Effects of Binding Site Mutations in Calmodulin.

Despite decades of research on ion-sensing proteins, gaps persist in the understanding of ion binding affinity and selectivity even in well-studied proteins such as calmodulin. Site-directed mutagenesis is a powerful and popular tool for addressing outstanding questions about biological ion binding and is employed to selectively deactivate binding sites and insert chromophores at advantageous positions within ion binding structures. However, even apparently nonperturbative mutations can distort the binding dynamics they are employed to measure. We use Fourier transform infrared (FTIR) and ultrafast two-dimensional infrared (2D IR) spectroscopy of the carboxylate asymmetric stretching mode in calmodulin as a mutation- and label-independent probe of the conformational perturbations induced in calmodulin's binding sites by two classes of mutation, tryptophan insertion and carboxylate side-chain deletion, commonly used to study ion binding in proteins. Our results show that these mutations not only affect ion binding but also induce changes in calmodulin's conformational landscape along coordinates not probed by vibrational spectroscopy, remaining invisible without additional perturbation of binding site structure. Comparison of FTIR line shapes with 2D IR diagonal slices provides a clear example of how nonlinear spectroscopy produces well-resolved line shapes, refining otherwise featureless spectral envelopes into more informative vibrational spectra of proteins.

Lanthanides compete with calcium for binding to cadherins and inhibit cadherin-mediated cell adhesion.

Lanthanides are rare-earth metals with a broad range of applications in biological research and medicine. In addition to their unique magnetic and spectroscopic properties, lanthanides are also effective mimics of calcium and can stimulate or inhibit the function of calcium-binding proteins. Cadherins are a large family of calcium-binding proteins that facilitate cell adhesion and play key roles in embryo development, tissue homeostasis and tumour metastasis. However, whether lanthanides can bind cadherins and functionally replace calcium binding has not been comprehensively explored. In this study, we investigated the effect of lanthanide binding on cadherin structure and function using terbium, which is a commonly used lanthanide for protein spectroscopy and a proposed anti-cancer agent. We demonstrate that terbium can compete with calcium for binding to calcium-binding sites in cadherins. Terbium binding to cadherins abolished their cell adhesive activity and rendered cadherins sensitive to proteolysis by trypsin. Molecular dynamics simulations indicate that replacement of calcium by terbium results in structural rearrangements and increases the flexibility of the cadherin ectodomain. These changes in structure and dynamics are likely to underlie the inability of lanthanide-bound cadherins to support cell adhesion. Taken together, our findings further knowledge on lanthanide interactions with calcium-binding proteins and provide new insight into the influence of metal chemistry on cadherin structure, dynamics and function.

Terbium chloride influences Clostridium difficile spore germination.

The germination of Clostridium difficile spores is an important stage of the C. difficile life cycle. In other endospore-forming bacteria, the composition of the medium in which the spores are generated influences the abundance of germination-specific proteins, thereby influencing the sensitivity of the spores towards germinants. In C. difficile media composition on the spores has only been reported to influence the number of spores produced. One of the measures of spore germination is the analysis of the release of DPA from the spore core. To detect DPA release in real time, terbium chloride is often added to the germination conditions because Tb3+ complexes with the released DPA and this can be detected using fluorescence measurements. Although C. difficile spores germinate in response to TA and glycine, recently calcium was identified as an enhancer for spore germination. Here, we find that germination by spores prepared in peptone rich media, such as 70:30, is positively influenced by terbium. We hypothesize that, in these assays, Tb3+ functions similarly to calcium. Although the mechanism(s) causing increased sensitivity of the C. difficile spores that are prepared in peptone rich media to terbium is still unknown, we suggest that the TbCl3 concentration used in the analysis of C. difficile DPA release be carefully titrated so as not to misinterpret future findings.

N­(6­Aminohexyl)­5­chloro­1­naphthalenesulfonamide, a centrin antagonist, inhibits Tb3+/peptides-binding properties.

N­(6­Aminohexyl)­5­chloro­1­naphthalenesulfonamide (W-7), a kind of adjuvant chemotherapy, can bind to calmodulin and inhibit Ca2+/calmodulin-regulated enzyme activities and cell proliferation. Similar to calmodulin, euplotes octocarinatus centrin (EoCen) belongs to EF-hand superfamily of calcium-binding proteins. It is associated with nucleotide excision repair (NER), cell division cycle and ciliogenesis. In the present study, the comparative interaction of W-7 with EoCen was first examined by using various spectroscopic, calorimetric methods and molecular docking. The obtain results recommend that only one W-7 molecule is identified binding to the C-terminal hydrophobic pocket of centrin that normally plays a role in anchoring targets. Methyl groups of Ala126, Met141, Ile161 and M162 of C-terminal may react with W-7 chloronaphthalene ring, other aliphatic or aromatic side-chains in a deep hydrophobic pocket of protein. Circular dichroism (CD) and fluorescence lifetime experiments reveal that W-7 triggers a conformational change of centrin. As a result, W-7 is identified to be an antagonist of centrin. It appears to inhibit the centrin-mediated activation of target proteins by blocking the hydrophobic pocket. Moreover, the complex formation leads to affinity decrease of Tb3+ binding to C-terminal of protein and self-assembly affected. Our present study provides the first view of centrin recognizing a naphthalene-sulfonamide derivative. It is proposed that W-7 and its analogues can serve as a useful tool for research on the participation of centrin in biological processes and cell biology-related studies.

Pore Engineering for Enhanced Mass Transport in Encapsulin Nanocompartments.

Encapsulins are robust and engineerable proteins that form hollow, nanosized, icosahedral capsids, making them attractive vehicles for drug delivery, scaffolds for synthetic bionanoreactors, and artificial organelles. A major limitation of native encapsulins is the small size of pores in the protein shell. At 3 Å diameter, these pores impose significant restrictions on the molecular weight and diffusion rate of potential substrates. By redesigning the pore-forming loop region in encapsulin from Thermotoga maritima, we successfully enlarged pore diameter up to an estimated 11 Å and increased mass transport rates by 7-fold as measured by lanthanide ion diffusion assay. Our study demonstrates the high tolerance of encapsulin for protein engineering and has created a set of novel, functionally improved scaffolds for applications as bionanoreactors.

Peptides as biosorbents - Promising tools for resource recovery.

Despite many innovations, meeting both economic and ecological requirements remains challenging for conventional resource recovery technology. The development of highly selective peptides puts a new competitor on the market. We present an approach to identify peptides for resource recovery using Phage Surface Display. Here, we describe the development of peptides for binding of rare earth element terbium-containing solids and for removal and enrichment of the heavy metal ions of cobalt and nickel out of waste waters and leaching solutions. We identified phage displaying specific peptides with ∼100× enhanced affinity towards terbium-containing solids or ∼20× enhanced affinity towards nickel (∼3× cobalt).

Modulation effect of double strand DNA on the self-assembly of N-terminal domain of Euplotes octocarinatus centrin.

Centrin is a member of the EF-hand super family of calcium-binding proteins, which can behave as a part of damage detector initiated the initiation of nucleotide excision repair (NER). Its self-assembly plays a causative role in fiber contraction associated with the cell division cycle and ciliogenesis. To explore the possible role of DNA in the process of centrin self-assembly, the aggregation properties of N-terminal domain of Euplotes octocarinatus centrin (N-EoCen) in the presence of DNA with or without metal ions are investigated. It is verified that metal ions, such as Ca2+ and Tb3+, can bind to N-EoCen with 2:1 stoichiometry by isothermal titration calorimetry (ITC). Importantly, this study reports that double strand DNA (dsDNA) is capable of binding N-EoCen, changing conformation of protein and modulating centrin aggregation, as demonstrated by extensive biophysical assays. Interestingly, the open conformation of protein induced by metal ions may be favour of the interaction of protein with dsDNA. Nevertheless, the randomly coiled single strand DNA (ssDNA) is completely inefficient to the aggregation regulation. Furthermore, results reveal that hydrophobic site could play important role in the process. This finding may link to the potent roles of centrin in the NER process.

Modulation of XPC peptide on binding Tb3+ to Euplotes octocarinatus centrin.

Centrins are Ca2+-binding proteins found throughout eukaryotic organisms. Xeroderma pigmentosum group C protein (XPC), a dominant component of the nuclear excision repair (NER) pathway, is a critical target protein of centrins. A 22-residue peptide (K842-R863) from XPC was used to investigate the effect of metal ions (Ca2+ and Tb3+) on the peptide binding of Euplotes octocarinatus centrin (EoCen) by isothermal titration calorimetry (ITC) and fluorescence spectroscopy. ITC and tryptophan spectrofluorimetric titrations revealed that metal ions (Ca2+ and Tb3+) could enhance the affinity between EoCen and the XPC peptide, and the enhanced effects were closely related to the ion potential of metal ions. Since the ion potential of Tb3+ (e/r = 0.0325) is larger than that of Ca2+ (e/r = 0.0202), the conformational change in the protein induced by Tb3+ is larger than that induced by Ca2+, and the enhanced affinity of Tb3+ is stronger than that of Ca2+. This interaction was driven by enthalpy in the presence of EDTA and enthalpy and entropy in the presence of Ca2+ or Tb3+. Similar to that observed in the presence of EDTA, the N-terminal domain did not participate in the interaction with the XPC peptide even in the presence of metal ions. Resonance light scattering (RLS) and the band shift in native polyacrylamide gel electrophoresis (PAGE) suggested that peptide binding resulted in the dissociation of EoCen aggregates and complex formation via the monomer-peptide form. Tb3+-Sensitized emission suggested that peptide binding in turn also had an impact on the Tb3+ binding of the protein: the C-terminal domain was slightly strengthened and the N-terminal domain was weakened about 225 fold. RLS and native PAGE indicated that the self-assembly induced by Tb3+ binding to the N-terminal domain of EoCen was inhibited in the presence of the XPC peptide. This study elucidates the molecular mechanism of EoCen function in the cellular context.