The classical simulated body fluids method cannot be employed to prepare biomimetic apatites encompassing metallic ions that lead to very stable phosphates. This is the case for heavy metals such as ...uranium, whose presence in bone mineral after contamination deserves toxicological study. We have demonstrated that existing methods, based on alternate dipping into calcium and phosphate ions solutions, can be adapted to achieve this aim. We have also especially studied the impact of the presence of carbonate ions in the medium as these are necessary to avoid hydrolysis of the contaminating metallic cations. Both the apatite-collagen complex method and a standard chemical (STD) method employing only mineral solutions lead to biomimetic apatites when calcium and carbonate ions are introduced simultaneously. The obtained materials were fully characterized and we established that the STD method tolerates the presence of carbonate ions much better, and this leads to homogeneous samples. Emphasis was set on the repeatability of the method to ensure the relevancy of further work performed on series of samples. Finally, osteoblasts cultured on these samples also proved a similar yield and standard-deviation in their adenosine triphosphate content when compared to commercially available substrates designed to study of such cell cultures.
Uranium has been shown to interfere with bone physiology and it is well established that this metal accumulates in bone. However, little is known about the effect of natural uranium on the behavior ...of bone cells. In particular, the impact of uranium on osteoclasts, the cells responsible for the resorption of the bone matrix, is not documented. To investigate this issue, we have established a new protocol using uranyl acetate as a source of natural uranium and the murine RAW 264.7 cell line as a model of osteoclast precursors. Herein, we detailed all the assays required to test uranium cytotoxicity on osteoclast precursors and to evaluate its impact on the osteoclastogenesis and on the resorbing function of mature osteoclasts. The conditions we have developed, in particular for the preparation of uranyl-containing culture media and for the seeding of RAW 264.7 cells allow to obtain reliable and highly reproductive results. Moreover, we have optimized the use of software tools to facilitate the analysis of various parameters such as the size of osteoclasts or the percentage of resorbed matrix.
To improve general understanding of biochemical mechanisms in the field of uranium toxicology, the identification of protein targets needs to be intensified. Immobilized metal affinity chromatography ...(IMAC) has been widely developed as a powerful tool for capturing metal binding proteins from biological extracts. However uranyl cations (UO
2
2+) have particular physico-chemical characteristics which prevent them from being immobilized on classical metal chelating supports. We report here on the first development of an immobilized uranyl affinity chromatography method, based on the cation-exchange properties of aminophosphonate groups for uranyl binding. The cation distribution coefficient and loading capacity on the support were determined. Then the stability of the uranyl-bonded phase under our chromatographic conditions was optimized to promote affinity mechanisms. The successful enrichment of uranyl binding proteins from human serum was then proven using proteomic and mass spectral analysis.
The impact of actinides on living organisms has been the subject of numerous studies since the 1950s. From a general point of view, these studies show that actinides are chemical poisons as well as ...radiological hazards. Actinides in plasma are assumed to be mainly complexed to transferrin, the iron carrier protein. This paper casts light on the uptake of actinides(IV) (thorium, neptunium, plutonium) by transferrin, focusing on the pH dependence of the interaction and on a molecular description of the cation binding site in the protein. Their behavior is compared with that of iron(III), the endogenous transferrin cation, from a structural point of view. Complementary spectroscopic techniques (UV/Vis spectrophotometry, microfiltration coupled with γ spectrometry, and X‐ray absorption fine structure) have been combined in order to propose a structural model for the actinide‐binding site in transferrin. Comparison of our results with data available on holotransferrin suggests some similarities between the behavior of FeIII and NpIV/PuIV/ NpIV is not complexed at pH <7, whereas at pH ≈7.4 complexation can be regarded as quantitative. This pH effect is consistent with the in vivo transferrin “cycle”. PuIV also appears to be quantitatively bound by apotransferrin at around pH ∼7.5, whereas ThIV was never complexed under our experimental conditions. EXAFS data at the actinide edge have allowed a structural model of the actinide binding site to be elaborated: at least one tyrosine residue could participate in the actinide coordination sphere (two for iron), forming a mixed hydroxo–transferrin complex in which actinides are bound with transferrin both through An–tyrosine and through AnOH bonds. A description of interatomic distances is provided.
A protein binds actinides: Transferrin (Tf), the iron‐transport protein in the serum, has been shown to take up neptunium and plutonium at oxidation state +IV. A molecular description of the actinide binding site has been proposed on the basis of several techniques, among them EXAFS (see figure).
We have designed new double stimuli-responsive polyelectrolyte microcapsules to be useable under physiological conditions to handle biomacromolecules while avoiding the risk of denaturation. They are ...made of poly(4-vinylpyridine hydrochloride) (PVP) and poly(sodium styrene sulfonate) (PSS). The microcontainers are sensitive to temperature variation, as they irreversibly shrink under heating. In addition, the capsules reversibly swell at pH > 6, making it possible to encapsulate human serum proteins by diffusion through the polymer membrane. Encapsulation efficiency is quantified by fluorescence techniques.
Nanoparticles (NPs) entering a biological fluid undergo surface modification due to dynamic, physicochemical interactions with biological components, especially proteins. In this work we used ...complementary bio-physico-chemical approaches to characterize the effects of interactions between CeO
2
NPs, immunoglobulins (IgGs) and bovine serum albumin (BSA) of a similar size on protein structural evolution as well as formation of (hetero-) aggregates. Using circular dichroism we showed that IgGs and BSA underwent significant structural changes after interaction with NPs. The NPs and protein-NPs were observed after size exclusion chromatography, highlighting the fact that few aggregates were stable enough to pass this mild separation step. X-ray absorption spectroscopy suggested that the surface chemistry of NPs was not affected by these proteins, also implying weak interactions. Competitive experiments revealed that, while the serum was more concentrated for BSA, IgG-NP aggregates were more stable. Altogether, our results indicate that, under our experimental conditions, the formation of a "protein corona" is an unstable and reversible mechanism. This indicates that, when NPs and proteins are similar in size, the adsorption concept (
i.e.
protein corona concept) cannot be applied to model the NP-protein interaction, and a heteroaggregation model is more appropriate.
This work demonstrates that a heteroaggregation model is more applicable than the adsorption concept when NPs and proteins have similar sizes.
Bacteria of the genus
Photobacterium
thrive worldwide in oceans and show substantial eco-physiological diversity including free-living, symbiotic and piezophilic life styles. Genomic characteristics ...underlying this variability across species are poorly understood. Here we carried out genomic and physiological analysis of
Photobacterium phosphoreum
strain ANT-2200, the first deep-sea luminous bacterium of which the genome has been sequenced. Using optical mapping we updated the genomic data and reassembled it into two chromosomes and a large plasmid. Genomic analysis revealed a versatile energy metabolic potential and physiological analysis confirmed its growth capacity by deriving energy from fermentation of glucose or maltose, by respiration with formate as electron donor and trimethlyamine
N
-oxide (TMAO), nitrate or fumarate as electron acceptors, or by chemo-organo-heterotrophic growth in rich media. Despite that it was isolated at a site with saturated dissolved oxygen, the ANT-2200 strain possesses four gene clusters coding for typical anaerobic enzymes, the TMAO reductases. Elevated hydrostatic pressure enhances the TMAO reductase activity, mainly due to the increase of isoenzyme TorA1. The high copy number of the TMAO reductase isoenzymes and pressure-enhanced activity might imply a strategy developed by bacteria to adapt to deep-sea habitats where the instant TMAO availability may increase with depth.
Vibrational spectroscopy gives important information on the properties of ligand and metal–ligand bonds in metalloenzymes. Infrared spectroscopy is appealing for the study of metal active sites that ...are not amenable to Raman spectroscopy. We present a combined experimental and theoretical approach to analyze the mid‐ and far‐IR spectra of Cu,Zn‐superoxide dismutase (Cu,Zn‐SOD) as a probe of the histidine ligands. This metalloenzyme provides a unique model to identify specific IR signatures of metal–histidine coordination and to study their alterations as a function of the metal (copper/zinc), the copper valence state (+I/+II), the histidine coordination mode (Nτ and Nπ) and the histidine protonation state. DFT calculations combined with normal mode descriptions from potential energy distribution calculations were performed on two slightly different cluster models. Differences in the constraints at the side chain of one histidine Cu ligand sensibly modify the geometric parameters and vibrational properties. Electrochemically induced FTIR difference spectroscopy provided mid‐ and far‐IR fingerprint spectra of the Cu protein in aqueous media that are sensitive to the redox state of the Cu centre at the active site. Comparisons of the DFT predictions with the experimental IR modes of the histidine ligands at the Cu,Zn‐SOD active site showed that useful mid‐IR markers of histidine Nτ and Nπ coordination were predicted with good accuracy. The DFT analysis further demonstrated a link between the ν(C4–C5) mode frequency of His46 and the specific properties of the His46–Cu bond in Cu,Zn‐SOD. A combined theoretical and experimental approach on samples in H2O and 2H2O or 15N‐labelled samples identified the contributions from the histidine side chain modes in the 669–629 cm–1 region.
The metal–histidine vibrations of the Cu,Zn‐superoxide dismutase enzyme are analyzed by mid‐ and far‐infrared difference spectroscopy. The histidine metal–ligand markers are analyzed and assigned from a combination of experimental and computational data. The contributions of the histidine modes are identified through additional data collected and calculated for labelled samples.
Abstract
The uranyl cation (UO
2
2+
) can be suspected to interfere with the binding of essential metal cations to proteins, underlying some mechanisms of toxicity. A dedicated computational screen ...was used to identify UO
2
2+
binding sites within a set of nonredundant protein structures. The list of potential targets was compared to data from a small molecules interaction database to pinpoint specific examples where UO
2
2+
should be able to bind in the vicinity of an essential cation, and would be likely to affect the function of the corresponding protein. The C‐reactive protein appeared as an interesting hit since its structure involves critical calcium ions in the binding of phosphorylcholine. Biochemical experiments confirmed the predicted binding site for UO
2
2+
and it was demonstrated by surface plasmon resonance assays that UO
2
2+
binding to CRP prevents the calcium‐mediated binding of phosphorylcholine. Strikingly, the apparent affinity of UO
2
2+
for native CRP was almost 100‐fold higher than that of Ca
2+
. This result exemplifies in the case of CRP the capability of our computational tool to predict effective binding sites for UO
2
2+
in proteins and is a first evidence of calcium substitution by the uranyl cation in a native protein.
Abstract The uranyl cation (UO$_2$$^{2+}$) can be suspected to interfere with the binding of essential metal cations to proteins, underlying some mechanisms of toxicity. A dedicated computational ...screen was used to identify UO$_2$$^{2+}$ binding sites within a set of nonredundant protein structures. The list of potential targets was compared to data from a small molecules interaction database to pinpoint specific examples where UO$_2$$^{2+}$ should be able to bind in the vicinity of an essential cation, and would be likely to affect the function of the corresponding protein. The C‐reactive protein appeared as an interesting hit since its structure involves critical calcium ions in the binding of phosphorylcholine. Biochemical experiments confirmed the predicted binding site for UO$_2$$^{2+}$ and it was demonstrated by surface plasmon resonance assays that UO$_2$$^{2+}$ binding to CRP prevents the calcium‐mediated binding of phosphorylcholine. Strikingly, the apparent affinity of UO$_2$$^{2+}$ for native CRP was almost 100‐fold higher than that of Ca$^{2+}$ . This result exemplifies in the case of CRP the capability of our computational tool to predict effective binding sites for UO$_2$$^{2+}$ in proteins and is a first evidence of calcium substitution by the uranyl cation in a native protein.
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