The titanium binding protein of Rhodococcus ruber GIN1 (NCIMB 40340) is a cell-surface homolog of the cytosolic enzyme dihydrolipoamide dehydrogenase

Rhodococcus ruber GIN1 (formally Rh. strain GIN1) was previously isolated on the basis of its strong adherence to coal fly ash (CFA) and titanium dioxide particles from CFA sedimentation ponds of an electrical power plant in Israel. The interaction of the bacterium with oxides has been shown to be mediated by a cell surface protein designated TiBP (titanium binding protein) involving primarily strong, non-electrostatic forces. In this work, we set forward to identify this unique exocellular protein. Sequence analysis of the purified protein by mass spectrometry (LC/MS/MS) following trypsinization revealed 11 peptides. All of them showed >90% amino acid residues identity with sequences of one of the orthologs (dldh1) of the cytosolic enzyme dihydrolipoamide dehydrogenase (DLDH), based on the genome sequence of Rhodococcus strain RHA1. This genome was selected as a reference since currently it is the only sequenced Rhodococcal genome. Altogether, these peptides covered over 25% of the 52 kDa protein molecule. N- and C-termini primers were prepared and used to sequence the paralog gene from Rh. ruber GIN1 after polymerase chain reaction (PCR) amplification. All 11 peptides showed 100% identity with the sequence of this gene. The homology of TiBP with the supposedly cytosolic DLDH raised the question of whether the exocellular TiBP possesses DLDH activity. Indeed, intact late logarithmic phase Rh. ruber GIN1 cells, previously shown to express TiBP, were found to possess such activity, while very low activity was associated with stationary phase cells which possess diminished TiBP expression on their surface. Further evidence for the exocellular location of TiBP/DLDH was achieved using specific anti-TiBP polyclonal antibodies by whole cell and protein enzyme-linked immunosorbent assay (ELISA), showing high reactivity of the logarithmic phase cell surface and substantially lower reactivity with the stationary phase cells. As expected, logarithmic phase spheroplasts were not recognized by these antibodies. Similar results were obtained by fluorescence and scanning electron microscopy. Our postulation that DLDH is located on the surface of Rh. ruber GIN1, serving as a TiO2 binding protein, is in accordance with literary evidence on DLDH in other organisms, Bacteria, Archea, and Eukaryots that suggests it is associated with the outer membranes or cell surfaces. As an exocellular protein DLDH assumes various tasks which are not related to its classical role as a 2-oxoacid dehydrogenase, including serving as an adhesion/binding protein in certain bacteria.     

Dihydrolipoamide dehydrogenase moonlighting activity as a DNA chelating agent

Dihydrolipoamide dehydrogenase (DLDH) is a mitochondrial enzyme that comprises an essential component of the pyruvate dehydrogenase complex. Lines of evidence have shown that many dehydrogenases possess unrelated actions known as moonlightings in addition to their oxidoreductase activity. As part of these activities, we have demonstrated that DLDH binds TiO₂ as well as produces reactive oxygen species (ROS). This ROS production capability was harnessed for cancer therapy via integrin‐mediated drug‐delivery of RGD‐modified DLDH (DLDH^RGD), leading to apoptotic cell death. In these experiments, DLDH^RGD not only accumulated in the cytosol but also migrated to the cell nuclei, suggesting a potential DNA‐binding capability of this enzyme. To explore this interaction under cell‐free conditions, we have analyzed DLDH binding to phage lambda (λ) DNA by gel‐shift assays and analytic ultracentrifugation, showing complex formation between the two, which led to full coverage of the DNA molecule with DLDH molecules. DNA binding did not affect DLDH enzymatic activity, indicating that there are neither conformational changes nor active site hindering in DLDH upon DNA‐binding. A Docking algorithm for prediction of protein‐DNA complexes, Paradoc, identified a putative DNA binding site at the C‐terminus of DLDH. Our finding that TiO₂‐bound DLDH failed to form a complex with DNA suggests partial overlapping between the two sites. To conclude, DLDH binding to DNA presents a novel moonlight activity which may be used for DNA alkylating in cancer treatment.     

Exfoliated oral mucosa cells as bioindicators of short- and long-term systemic titanium contamination

BackgroundHumans are exposed to exogenous sources of titanium-containing particles that can enter the body mainly by inhalation, ingestion, or dermal absorption. Given the widespread use of biomaterials in medicine, the surface of a titanium (Ti) biomedical device is a potential endogenous source of Ti ions and/or Ti-containing particles, such as TiO₂ micro-(MPs) and nano-particles (NPs), resulting from biotribocorrosion processes. Ti ions or Ti-containing particles may deposit in epithelial cells of the oral mucosa, and the latter may therefore serve as bioindicators of short and long-term systemic Ti contamination. The aim of the present study was to histologically and quantitatively evaluate the presence of Ti traces in cells exfoliated from the oral mucosa as possible bioindicators of systemic contamination with this metal at short and long-term experimental time pointsMethodsThirty Wistar rats were intraperitoneally injected with a suspension of titanium dioxide (TiO₂) (0.16 g/100 g body weight of TiO₂ in 5 ml of NaCl 0.9%) using 5 nm NPs (Group: TiO₂-NP5; n = 10), 45 µm MPs (Group: TiO₂-MP45; n = 10), or vehicle alone (Control group; n = 10). At one and six months post-injection, right-cheek mucosa cells were obtained by exfoliative cytology using a cytobrush; they were spray fixed and stained using Safranin or the Papanicolaou technique. The smears were cytologically evaluated (light microscopy) to determine the presence of particulate material, which was also analyzed microchemically (SEM-EDS). Left-cheek mucosa cells were similarly obtained and re-suspended in 5 ml of PBS (pH: 7.2–7.4); the samples corresponding to each group were pooled together and analyzed spectrometrically (ICP-MS) to determine Ti concentration in each of the studied groups. Blood samples were obtained for histological determination of the presence of particulate material on Safranin-stained blood smears and determination of plasma concentration of Ti by ICP-MSResultsDifferent size and shape metal-like particles were observed inside and outside epithelial cells in TiO₂-NP5 and TiO₂-MP45 cytological smears at both one and six months post-injection. EDS analysis showed the presence of Ti in the particles. ICP-MS revealed higher Ti concentrations in both TiO₂ injected groups compared to the control group. In addition, Ti concentration did not vary with time or particle size. Monocytes containing particles were observed in blood smears of TiO₂-exposed animals one- and six-months post-injection. Plasma levels of Ti were significantly higher in TiO₂-NP5- and TiO₂-MP45- exposed animals than in controls (p < 0.05), and Ti concentration was significantly higher at one month than at six months in both TiO₂-exposed groups (p < 0.05).ConclusionsCells exfoliated from the oral mucosa could be used as bioindicators of short- and long-term systemic contamination with Ti. Exfoliative cytology could be used as a simple, non-invasive, and inexpensive diagnostic method for monitoring biotribocorrosion of Ti implants and patient clinical follow-up.     

Cellular Toxicity of TiO2 Nanoparticles in Anatase and Rutile Crystal Phase

Titanium dioxide nanoparticles are massively produced and widely used in daily life, which has posed potential risk to human health. However, the molecular mechanism of TiO₂ nanoparticles (NPs) with different crystal phases is not clear. In this study, the characterization of two crystalline phases of TiO₂ NPs is evaluated by transmission electron microscopy and X-ray absorption fine structure spectrum; an interaction of these TiO₂ NPs with HaCaT cells is studied in vitro using transmission electron microscopy, chemical precipitation method, and X-ray absorption fine structure spectrometry. The coordination and surface properties indicate that only the anatase–TiO₂ NPs allow spontaneous reactive oxygen species (ROS) generation, but rutile–TiO₂ NPs do not after dispersion. The interaction between TiO₂ NPs and cellular components might also generate ROS for both anatase–TiO₂ NPs and rutile–TiO₂ NPs. The ROS generation could lead to cellular toxicity if the level of ROS production overwhelms the antioxidant defense of the cell or induces the mitochondrial apoptotic mechanisms. Furthermore, Ti had a direct combination with some protein or DNA after NPs enter the cell, which could also lead to cellular toxicity.     

Antibacterial effect of hydrogen peroxide-titanium dioxide suspensions in the decontamination of rough titanium surfaces

The chemical decontamination of infected dental implants is essential for the successful treatment of peri-implantitis. The aim of this study was to assess the antibacterial effect of a hydrogen peroxide-titanium dioxide (H₂O₂–TiO₂) suspension against Staphylococcus epidermidis biofilms. Titanium (Ti) coins were inoculated with a bioluminescent S. epidermidis strain for 8 h and subsequently exposed to H₂O₂ with and without TiO₂ nanoparticles or chlorhexidine (CHX). Bacterial regrowth, bacterial load and viability after decontamination were analyzed by continuous luminescence monitoring, live/dead staining and scanning electron microscopy. Bacterial regrowth was delayed on surfaces treated with H₂O₂–TiO₂ compared to H₂O₂. H₂O₂-based treatments resulted in a lower bacterial load compared to CHX. Few viable bacteria were found on surfaces treated with H₂O₂ and H₂O₂–TiO₂, which contrasted with a uniform layer of dead bacteria for surfaces treated with CHX. H₂O₂–TiO₂ suspensions could therefore be considered an alternative approach in the decontamination of dental implants.     

Titanium preferential binding sites in human serum transferrin at physiological concentrations

Serum transferrin (Tf) is an iron binding glycoprotein that plays a central role in the metabolism of this essential metal but it also binds other metal ions. Four main transferrin forms containing different iron binding states can be distinguished in human serum samples: monoferric (C-site or N-site), holotransferrin (with two Fe atoms) and apotransferrin (with no metal). Recently, it has been reported that Tf binds also Ti even more tightly than does Fe, in artificially Ti(iv) spiked solutions. However, very limited work has been done on the Ti binding to Tf at physiological concentrations in patients carrying intramedullary Ti nails. Here we report the chemical association of Ti to Tf "in vivo" under different chromatographic conditions by elemental mass spectrometry using double focusing inductively coupled plasma (DF-ICP-MS) as detector. For the separation of the Ti/Fe-Tf forms different gradient conditions have been explored. The observed results reveal that human serum Ti (from patients carrying intramedullary Ti nails) is uniquely associated to the N-lobe of Tf. The investigation of the influence of sialic acid in the carbohydrate chain of human serum Tf, studied by incubating the protein with neuraminidase (sialidase) to obtain the monosialilated species, revealed that the binding affinity of Ti was similar for monosialo-Tf and for native-Tf and occurs in the N-lobe. These results suggest that the species Fe(C)Ti(N)-TF might provide a route for Ti entry into cells via the transferrin receptors after the release of the metal from its implants.     

Mesoporous TiO2 nanoparticles: A new material for biolistic bombardment

To date, only solid heavy metals such as gold or tungsten have been used as DNA carriers in biolistic bombardment of algae. In this study, we show that even a metal oxide of lower density can act as a DNA carrier. We investigated the potency of size‐controlled mesoporous titanium dioxide (TiO₂) particles. Among the six tested gas pressures, TiO₂ particles best facilitated transformation of the green alga Chlamydomonas reinhardtii at 1100 psi (approximately 7.6 MPa) and 2000 psi (approximately 14 MPa). Surprisingly, a mesoporous metal oxide with a density of approximately only one‐tenth that of gold or tungsten could be effective as a DNA carrier in biolistic bombardment of a rigid cell wall‐containing alga. In addition, we found two peaks of gas pressures in the transformation ratio irrespective of whether the particles were made of gold, tungsten, or TiO₂.     

Proteomic analysis of bone proteins adsorbed onto the surface of titanium dioxide

Osseointegration is the structural and functional connection between bone tissues and implants such as titanium dioxide (TiO₂). The bone-TiO₂ interface is thought to contain proteoglycans. However, exhaustive analysis of the proteins in this layer has not been performed. In this study, we evaluated the bone protein adhered on the surface of TiO₂ comprehensively. Pig bone protein was extracted by sequential elutions with guanidine, 0.1 M EDTA, and again with guanidine. The proteins obtained from these extractions were allowed to adhere to an HPLC column packed with TiO₂ and were eluted with 0.2 M NaOH. The eluted proteins were identified by LC/MS/MS and included not only proteoglycans but also other proteins such as extracellular matrix proteins, enzymes, and growth factors. Calcium depositions were observed on TiO₂ particles incubated with bone proteins, guanidine-extracted proteins adhered to TiO₂ displayed significantly high amounts of calcium depositions.     

Prophylactic administration of carnosine and melatonin abates the incidence of renal toxicity induced by an over dose of titanium dioxide nanoparticles

The alleviative effects of two antioxidants, carnosine (Car) and melatonin (Mel), against titanium dioxide nanoparticles (TiO₂‐NPs) toxicity‐induced oxidative and inflammatory renal damage were examined in rats. Administration of these antioxidants along with TiO₂‐NPs effectively reduced serum urea, uric acid, creatinine, glucose, tumor necrosis factor‐α, interleukin‐6, C‐reactive protein, immunoglobulin G, vascular endothelial growth factor, and nitric oxide, as well as a significant amelioration of the decrease in glutathione levels in renal tissue was observed, compared to those in rats treated with TiO₂‐NPs alone. The renoprotective properties of the antioxidants were confirmed by reduced intensity of renal damage as demonstrated by histological findings. In conclusion, Car and Mel play protective roles against TiO₂‐NPs‐induced renal inflammation and oxidative injury, likely due to their antioxidant and anti‐inflammatory properties.     

Samarium doped titanium dioxide nanoparticles as theranostic agents in radiation therapy

PurposeTitanium dioxide nanoparticles (TiO₂ NPs) have been investigated for their role as radiosensitisers for radiation therapy. The study aims to increase the efficiency of these NPs by synthesising them with samarium.MethodsSamarium-doped TiO₂ NPs (Ti(Sm)O₂ NPs) were synthesised using a solvothermal method. Transmission electron microscopy (TEM), X-ray diffraction (XRD), and energy-dispersive X-ray spectroscopy (EDS) were performed for characterising of the Ti(Sm)O₂ NPs. The intracellular uptake and cytotoxicity were assessed in vitro using A549 and DU145 cancer cell lines. Furthermore, the effect of dose enhancement and generation of reactive oxygen species (ROS) in response to 6 MV X-rays was evaluated. Additionally, the image contrast properties were investigated using computed tomography (CT) images.ResultsThe synthesised Ti(Sm)O₂ NPs were about 13 nm in diameter as determined by TEM. The XRD pattern of Ti(Sm)O₂ NPs was consistent with that of anatase-type TiO₂. EDS confirmed the presence of samarium in the nanoparticles. At 200 μg/ml concentration, no differences in cellular uptake and cytotoxicity were observed between TiO₂ NPs and Ti(Sm)O₂ NPs in both A549 and DU145 cells. However, the combination of Ti(Sm)O₂ NPs and X-rays elicited higher cytotoxic effect and ROS generation in the cells than that with TiO₂ NPs and X-rays. The CT numbers of Ti(Sm)O₂ NPs were systematically higher than that of TiO₂ NPs.ConclusionsThe Ti(Sm)O₂ NPs increased the dose enhancement of MV X-ray beams than that elicited by TiO₂ NPs. Samarium improved the efficiency of TiO₂ NPs as potential radiosensitising agent.     

The Effects of Nano-anatase TiO2 on the Activation of Lactate Dehydrogenase from Rat Heart

Lactate dehydrogenase (LDH, EC1.1.1.27), widely expressed in the heart, liver, and other tissues, plays an important role in glycolysis and glyconeogenesis. The activity of LDH is often altered upon inflammatory responses in animals. Nano-TiO₂ was shown to provoke various inflammatory responses both in rats and mice; however, the molecular mechanism by which TiO₂ exerts its toxicity has not been completely understood. In this report, we investigated the mechanisms of nano-anatase TiO₂ (5 nm) on LDH activity in vitro. Our results showed that LDH activity was greatly increased by low concentration of nano-anatase TiO₂, while it was decreased by high concentration of nano-anatase TiO₂. The spectroscopic assays revealed that the nano-anatase TiO₂ particles were directly bound to LDH with mole ratio of [nano-anatase TiO₂] to [LDH] was 0.12, indicating that each Ti atom was coordinated with five oxygen/nitrogen atoms and a sulfur atoms of amino acid residues with the Ti–O(N) and Ti–S bond lengths of 1.79 and 2.41 Å. We postulated that the bound nano-anatase TiO₂ altered the secondary structure of LDH, created a new metal ion-active site for LDH, and thereby enhanced LDH activity.     

Titanium dioxide nanoparticles preferentially bind in subdomains IB,IIA of HSA and minor groove of DNA

Titanium dioxide nanoparticles (TiO₂-NPs) interaction with human serum albumin (HSA) and DNA was studied by UV–visible spectroscopy, spectrofluorescence, circular dichroism (CD), and transmission electron microscopy (TEM) to analyze the binding parameters and protein corona formation. TEM revealed protein corona formation on TiO₂-NPs surface due to adsorption of HSA. Intrinsic fluorescence quenching data suggested significant binding of TiO₂-NPs (avg. size 14.0 nm) with HSA. The Stern–Volmer constant (K_sv) was determined to be 7.6 × 10² M^?1 (r² = 0.98), whereas the binding constant (K_a) and number of binding sites (n) were assessed to be 5.82 × 10² M^?1 and 0.97, respectively. Synchronous fluorescence revealed an apparent decrease in fluorescence intensity with a red shift of 2 nm at Δλ = 15 nm and Δλ = 60 nm. UV–visible analysis also provided the binding constant values for TiO₂-NPs–HSA and TiO₂-NPs-DNA complexes as 2.8 × 10² M^?1 and 5.4 × 10³ M^?1. The CD data demonstrated loss in α-helicity of HSA and transformation into β-sheet, suggesting structural alterations by TiO₂-NPs. The docking analysis of TiO₂-NPs with HSA revealed its preferential binding with aromatic and non-aromatic amino acids in subdomain IIA and IB hydrophobic cavity of HSA. Also, the TiO₂-NPs docking revealed the selective binding with A-T bases in minor groove of DNA.     

Design and synthesis of magnetic binary metal oxides nanocomposites through dopamine chemistry for highly selective enrichment of phosphopeptides

In this work, for the first time, magnetic binary metal oxides nanocomposites which integrated Zr and Ti into one entity on an atomic scale on polydopamine coated magnetic graphene (magG/PD/(Zr‐Ti)O₄) was designed and synthesized, and applied to the enrichment of phosphopeptides. The newly prepared magG/PD/(Zr‐Ti)O₄ composites gathered the advantages of large surface area, superparamagnetism, biocompatibility and the enhanced affinity properties to phosphopeptides. MagG/PD/ZrO₂, magG/PD/TiO₂, as well as the simple physical mixture of them were introduced to compare with magG/PD/(Zr‐Ti)O₄ composites. High sensitivity (1 pg/μL or 4.0 × 10^–11 M) and selectivity (weight ratio of β‐casein and BSA reached up to 1:8000) toward phosphopeptides were also presented for magG/PD/(Zr‐Ti)O₄ composites. Additionally, mouse brain tissue was chose as the real samples to further investigate the phosphopeptides enrichment ability of this new material.     

UPTAKE AND TRANSLOCATION OF TI FROM NANOPARTICLES IN CROPS AND WETLAND PLANTS

Bioavailability of engineered metal nanoparticles affects uptake in plants, impacts on ecosystems, and phytoremediation. We studied uptake and translocation of Ti in plants when the main source of this metal was TiO₂ nanoparticles. Two crops (Phaseolus vulgaris (bean) and Triticum aestivum (wheat)), a wetland species (Rumex crispus, curly dock), and the floating aquatic plant (Elodea canadensis, Canadian waterweed), were grown in nutrient solutions with TiO₂ nanoparticles (0, 6, 18 mmol Ti L^?1 for P. vulgaris, T. aestivum, and R. crispus; and 0 and 12 mmol Ti L^?1 for E. canadensis). Also examined in E. canadensis was the influence of TiO₂ nanoparticles upon the uptake of Fe, Mn, and Mg, and the influence of P on Ti uptake. For the rooted plants, exposure to TiO₂ nanoparticles did not affect biomass production, but significantly increased root Ti sorption and uptake. R. crispus showed translocation of Ti into the shoots. E. canadensis also showed significant uptake of Ti, P in the nutrient solution significantly decreased Ti uptake, and the uptake patterns of Mn and Mg were altered. Ti from nano-Ti was bioavailable to plants, thus showing the potential for cycling in ecosystems and for phytoremediation, particularly where water is the main carrier.     

Comparative in vitro study regarding the biocompatibility of titanium-base composites infiltrated with hydroxyapatite or silicatitanate

Background

The development of novel biomaterials able to control cell activities and direct their fate is warranted for engineering functional bone tissues. Adding bioactive materials can improve new bone formation and better osseointegration. Three types of titanium (Ti) implants were tested for in vitro biocompatibility in this comparative study: Ti6Al7Nb implants with 25% total porosity used as controls, implants infiltrated using a sol–gel method with hydroxyapatite (Ti HA) and silicatitanate (Ti SiO₂). The behavior of human osteoblasts was observed in terms of adhesion, cell growth and differentiation.

Results

The two coating methods have provided different morphological and chemical properties (SEM and EDX analysis). Cell attachment in the first hour was slower on the Ti HA scaffolds when compared to Ti SiO₂ and porous uncoated Ti implants. The Alamar blue test and the assessment of total protein content uncovered a peak of metabolic activity at day 8–9 with an advantage for Ti SiO₂ implants. Osteoblast differentiation and de novo mineralization, evaluated by osteopontin (OP) expression (ELISA and immnocytochemistry), alkaline phosphatase (ALP) activity, calcium deposition (alizarin red), collagen synthesis (SIRCOL test and immnocytochemical staining) and osteocalcin (OC) expression, highlighted the higher osteoconductive ability of Ti HA implants. Higher soluble collagen levels were found for cells cultured in simple osteogenic differentiation medium on control Ti and Ti SiO₂ implants. Osteocalcin (OC), a marker of terminal osteoblastic differentiation, was most strongly expressed in osteoblasts cultivated on Ti SiO₂ implants.

Conclusions

The behavior of osteoblasts depends on the type of implant and culture conditions. Ti SiO₂ scaffolds sustain osteoblast adhesion and promote differentiation with increased collagen and non-collagenic proteins (OP and OC) production. Ti HA implants have a lower ability to induce cell adhesion and proliferation but an increased capacity to induce early mineralization. Addition of growth factors BMP-2 and TGFβ1 in differentiation medium did not improve the mineralization process. Both types of infiltrates have their advantages and limitations, which can be exploited depending on local conditions of bone lesions that have to be repaired. These limitations can also be offset through methods of functionalization with biomolecules involved in osteogenesis.

     

Distinct functions of serial metal‐binding domains in the Escherichia coli P1B‐ATPase CopA

P_1B‐ATPases are among the most common resistance factors to metal‐induced stress. Belonging to the superfamily of P‐type ATPases, they are capable of exporting transition metal ions at the expense of adenosine triphosphate (ATP) hydrolysis. P_1B‐ATPases share a conserved structure of three cytoplasmic domains linked by a transmembrane domain. In addition, they possess a unique class of domains located at the N‐terminus. In bacteria, these domains are primarily associated with metal binding and either occur individually or as serial copies of each other. Within this study, the roles of the two adjacent metal‐binding domains (MBDs) of CopA, the copper export ATPase of Escherichia coli were investigated. From biochemical and physiological data, we deciphered the protein‐internal pathway of copper and demonstrate the distal N‐terminal MBD to possess a function analogous to the metallochaperones of related prokaryotic copper resistance systems, that is its involvement in the copper transfer to the membrane‐integral ion‐binding sites of CopA. In contrast, the proximal domain MBD2 has a regulatory role by suppressing the catalytic activity of CopA in absence of copper. Furthermore, we propose a general functional divergence of tandem MBDs in P_1B‐ATPases, which is governed by the length of the inter‐domain linker.     

Improved enrichment strategies for phosphorylated peptides on titanium dioxide using methyl esterification and pH gradient elution

Improvements to phosphopeptide enrichment protocols employing titanium dioxide (TiO₂) are described and applied to identification of phosphorylation sites on recombinant human cyclin-dependent kinase 2 (CDK2). Titanium dioxide binds phosphopeptides under acidic conditions, and they can be eluted under basic conditions. However, some nonphosphorylated peptides, particularly acidic peptides, bind and elute under these conditions as well. These nonphosphorylated peptides contribute significantly to ion suppression of phosphopeptides and also increase sample complexity. We show here that the conversion of peptide carboxylates to their corresponding methyl esters sharply reduces nonspecific binding, improving the selectivity for phosphopeptides, just as has been reported for immobilized metal affinity chromatography (IMAC) columns. We also present evidence that monophosphorylated peptides can be effectively fractionated from multiply phosphorylated peptides, as well as acidic peptides, via stepwise elution from TiO₂ using pH step gradients from pH 8.5 to pH 11.5. These approaches were applied to human CDK2 phosphorylated in vitro by yeast CAK1p in the absence of cyclin. We confirmed phosphorylation at T160, a site previously documented and shown to be necessary for CDK2 activity. However, we also discovered several novel sites of partial phosphorylation at S46, T47, T165, and Y168 when ion-suppressing nonphosphorylated peptides were eliminated using the new protocols.     

Subunit dissociation and metal binding by Escherichia coli apo-manganese superoxide dismutase

Metal binding by apo-manganese superoxide dismutase (apo-MnSOD) is essential for functional maturation of the enzyme. Previous studies have demonstrated that metal binding by apo-MnSOD is conformationally gated, requiring protein reorganization for the metal to bind. We have now solved the X-ray crystal structure of apo-MnSOD at 1.9 Å resolution. The organization of active site residues is independent of the presence of the metal cofactor, demonstrating that protein itself templates the unusual metal coordination geometry. Electrophoretic analysis of mixtures of apo- and (Mn₂)-MnSOD, dye-conjugated protein, or C-terminal Strep-tag II fusion protein reveals a dynamic subunit exchange process associated with cooperative metal binding by the two subunits of the dimeric protein. In contrast, (S126C) (SS) apo-MnSOD, which contains an inter-subunit covalent disulfide-crosslink, exhibits anti-cooperative metal binding. The protein concentration dependence of metal uptake kinetics implies that protein dissociation is involved in metal binding by the wild type apo-protein, although other processes may also contribute to gating metal uptake. Protein concentration dependent small-zone size exclusion chromatography is consistent with apo-MnSOD dimer dissociation at low protein concentration (K_D = 1 × 10⁻⁶ M). Studies on metal uptake by apo-MnSOD in Escherichia coli cells show that the protein exhibits similar behavior in vivo and in vitro.     

Efficient enrichment of phosphopeptides by magnetic TiO₂-coated carbon-encapsulated iron nanoparticles

Titanium dioxide (TiO₂) has been widely used for phosphopeptide enrichment. Several approaches have been reported to produce magnetic TiO₂ affinity probes. In this report, we present a facile approach to immobilize TiO₂ onto poly(acrylic acid)‐functionalized magnetic carbon‐encapsulated iron nanoparticles as affinity probes for efficient enrichment of phosphopeptides. By using the new magnetic TiO₂ affinity probes, denoted as TiO₂‐coated Fe@CNPs, rapid and effective MALDI‐TOF MS profiling of phosphopeptides was demonstrated in different model systems such as tryptic digests of β‐casein, and complex β‐casein/BSA mixture. The TiO₂‐coated Fe@CNPs out‐performed the commercial TiO₂‐coated magnetic beads for detection of phosphopeptides from tryptic digests of β‐casein/BSA mixture with a molar ratio of 1:100. The new TiO₂‐coated magnetic probes were also proven to be applicable for real life samples. The magnetic TiO₂‐coated Fe@CNPs were employed to selectively isolate phosphopeptides from tryptic digests of HeLa cell lysates and out‐performed the commercial magnetic TiO₂ beads in the number of identified phosphopeptides and phosphorylation sites. In a 200‐μg equivalent of HeLa cell lysates, we identified 1415 unique phosphopeptides and 1093 phosphorylation sites, indicating the good performance of the new approach.