Jean Philippe Renault

Structural Determinants for Protein adsorption/non-adsorption to Silica Surface

The understanding of the mechanisms involved in the interaction of proteins with inorganic surfaces is of major interest in both fundamental research and applications such as nanotechnology. However, despite intense research, the mechanisms and the structural determinants of protein/surface interactions are still unclear. We developed a strategy consisting in identifying, in a mixture of hundreds of soluble proteins, those proteins that are adsorbed on the surface and those that are not. If the two protein subsets are large enough, their statistical comparative analysis must reveal the physicochemical determinants relevant for adsorption versus non-adsorption. This methodology was tested with silica nanoparticles. We found that the adsorbed proteins contain a higher number of charged amino acids, particularly arginine, which is consistent with involvement of this basic amino acid in electrostatic interactions with silica. The analysis also identified a marked bias toward low aromatic amino acid content (phenylalanine, tryptophan, tyrosine and histidine) in adsorbed proteins. Structural analyses and molecular dynamics simulations of proteins from the two groups indicate that non-adsorbed proteins have twice as many π-π interactions and higher structural rigidity. The data are consistent with the notion that adsorption is correlated with the flexibility of the protein and with its ability to spread on the surface. Our findings led us to propose a refined model of protein adsorption.

Molecular cloning, expression analysis and iron metal cofactor characterisation of a superoxide dismutase from Toxoplasma gondii ☆

A genomic region of 12 kb encompassing the gene encoding the superoxide dismutase (SOD) of Toxoplasma gondii has been cloned. The gene contains four exons of 121, 42, 381 and 59 bp which are separated by three introns of 321, 202, and 577 bp, respectively. The open reading frame can be translated into a protein of 201 amino acids with a molecular mass of 22.6 kDa. Alignment indicated that it is a FeSOD, a type only found in bacteria, protozoa and chloroplast of higher plants. Recombinant SOD was expressed in a Escherichia coli double mutant lacking both MnFeSOD and FeSODs. The presence of iron as metal cofactor was confirmed by measurements of iron by absorption mass spectrometry and electron paramagnetic resonance studies. Semi-quantitative reverse transcribed polymerase chain reaction experiments showed a similar amount of SOD transcripts in two developmental stages of T. gondii. Antibodies raised against the purified recombinant protein detected SOD protein in both bradyzoite and tachyzoite forms suggesting this SOD might be essential for the intracellular growth of both developmental stages. Southern blot analysis indicated that SOD occured as a single copy gene in T. gondii genome.

High-throughput evaluation of antioxidant and pro-oxidant activities of polyphenols with thymidine protection assays

A recently reported high‐throughput screening strategy has been applied to the rapid selection of new water‐soluble antioxidants that display strong protective activities. Based on a competitive immunoassay, a triple‐screening procedure was used to evaluate the ability of different compounds to protect thymidine under different oxidative stresses. The pro‐oxidant effect of norbadione A in the presence of iron was observed, while some pulvinic acid derivatives proved strongly protective during γ radiolysis, UV irradiation, and Fenton‐like oxidation.

Myoglobin on Silica: A Case Study of the Impact of Adsorption on Protein Structure and Dynamics

If protein structure and function changes upon adsorption are well documented, modification of adsorbed protein dynamics remains a blind spot, despite its importance in biological processes. The adsorption of metmyoglobin on a silica surface was studied by isotherm measurements, microcalorimetry, circular dichroïsm, and UV-visible spectroscopy to determine the thermodynamic parameters of protein adsorption and consequent structure modifications. The mean square displacement and the vibrational densities of states of the adsorbed protein were measured by elastic and inelastic neutron scattering experiments. A decrease of protein flexibility and depletion in low frequency modes of myoglobin after adsorption on silica was observed. Our results suggest that the structure loss itself is not the entropic driving force of adsorption.

Solid-state NMR characterization of a controlled-pore glass and of the effects of electron irradiation.

Controlled-pore glasses (CPGs) are silica-based materials which provide an adequate model system for a better understanding of the radiation chemistry of glasses, especially under nanoscopic confinement. This paper presents a characterization of a nanoporous CPG before and after electron irradiation using multinuclear solid-state magnetic resonance (NMR). 1H MAS NMR has been used for studying the surface proton sites and it is observed that the irradiation leads to a dehydration of the material. Accordingly, concerning the silicon sites near the surface, the observed variation of the Q4, Q3 and Q2 species from 1H-29Si CPMAS spectra shows an increase of the surface polymerization under irradiation, implying in majority a Q2 to Q3/Q4 conversion mechanism. Similarly, 1H-17 O CPMAS measurements exhibit an increase of Si-O-Si groups at the expenses of Si-OH groups. In addition, modifications of the environment of the residual boron atoms are also put in evidence from 11B MAS and MQMAS NMR These data show that MAS NMR methods provide sensitive tools for the characterization of these porous glasses and of the tiny modifications occurring under electron irradiation.

A nanosecond pulse radiolysis study of the hydrated electron with high energy carbon ions

The radiolysis yields in pure water were determined using nanosecond pulse radiolysis using high linear energy transfer (LET) particles (1 ns pulses of high energy 1 GeV carbon ion beam). The main characteristics of this experiment were the nanosecond time resolution with heavy ion beam and the single value of LET along the ion track: 27 eV/nm. The kHz repetition rate of the pulsed beam used to reach a good signal-to-noise ratio in these experiments required the development of an acquisition method and a mathematical treatment of a DPO screen. The measured radiolytic yield for the hydrated electron at the nanosecond scale is quite high at 4.5×10−7 mol/J. A comparison of the experimental results obtained with a Monte Carlo simulation shows a rather good agreement at this LET value. To a certain extent this will allow a better adjustment of the Monte Carlo code.

H2 formation by electron irradiation of SBA-15 materials and the effect of CuII grafting

Measurement of H(2) production from electron irradiation (10 MeV) on SBA-15 materials has shown that adsorbed water is attacked preferentially. Silanol groups are only attacked when they are in the majority with respect to adsorbed water, however they are much less efficient at producing H(2). The comparison between water content before and after electron irradiation and the corresponding H(2) production indicates that water desorption is the main route to adsorbed water loss for SBA-15 materials. On the other hand, surface silanol groups are more susceptible to attack, leading to H(2) production when SBA-15 samples have undergone extensive thermal treatment. Electron irradiation of SBA-15-Cu materials has shown that the presence of Cu(II) on the surface reduces and inhibits the production of H(2.) This inhibiting power affects adsorbed water bonded to grafted copper but not surface silanol groups.

Ab initio study of Cd-thiol complexes: application to the modelling of the metallothionein active site

The metallothionein active site in vacuo was studied using ab initio methods as a necessary step in order to understand the relationship between the structure and the efficiency of the protein in binding cadmium ions. The natural Cd4Cys11 cluster located in the protein α-domain was represented by cadmium–thiolate complexes. First, it was shown, by comparison with available experimental results, that the geometry and the binding energy of cadmium coordination complexes can be correctly predicted at the RHF-MP2 theory level using the effective core potentials (ECP) and the double-ζ basis sets of Hay–Wadt, improved with polarization and diffuse functions. The computed geometry of the model system of the α-domain active site was then compared with that extracted from the X-ray crystal structure of the metallothionein. The significant difference between the two structures was attributed to the external constraints imposed in the natural cluster by the surrounding protein structure. The nature of the Cd–thiolate bond was further examined by calculating natural atomic populations and localizing the molecular orbitals. A good correlation between the Cd–S bond length and the amount of charge transferred from the ligand to the cadmium ion was found allowing an explanation of the effects of ligand protonation on the structure of mixed cadmium–thiol/thiolate complexes.

Finite Size Effects on Hydrogen Bonds in Confined Water

Bulk water has unique properties deriving from its ability to form a labile hydrogen-bond network. However, water is often not present in its bulk form but rather trapped in small cavities (e.g., water in concrete, clays, zeolites, and nanochannels). Consequently, many efforts have been devoted to describing the confinement effects on the structure and dynamics of water. The present understanding is that nanoconfinement induces a freezing of the molecular motions near the confining surface on a layer with a thickness of 0.4– 0.8 nm. Herein we clearly demonstrate that even large pores (up to 50 nm diameter), in which interfacial effects are negligible, can alter the properties of water. The hydrogenbond network properties in confined media have been traced in real time by transient absorption infrared spectroscopy of the OH vibration. We performed IR pump–probe experiments on nanoconfined water in fully hydrated controlled-pore glasses (CPG) of 1, 13, and 50 nm pore diameters, on surface water in low-hydration CPG (1 nm), and on bulk water. We decided to work with dilute HOD in D2O to suppress energy transfer between neighboring OH vibrators and to tune the optical density of the sample. Figure 1 shows the time dependence of the transient absorption anisotropy that is directly connected to the rotational diffusion of the water molecules. The characteristic rotational diffusion time increases when the pore size decreases. Such an evolution was to be expected as quasielastic neutron scattering studies have shown slower diffusive motions in small pores. For a pore size of 50 nm, the rotational time (2.8 ps) is almost identical to that measured in bulk water (2.5 ps). Water trapped in 1 nm pores compares well with surface water, for which the anisotropy, which remains high at long delay times, demonstrates hindered rotational motions. Similar behavior has been recently observed for water adsorbed both on soft and hard surfaces. All other transient absorption data (Figures 2B and 3) seem to contradict this apparent smooth dependence of the behavior of water on pore size. The transient spectra (Figure 2B) and the excited-state lifetime (Figure 3) show a discontinuous evolution when going from bulk to nanoconfined water and from nanoconfined to surface water. The transient spectra of water are equivalent (Figure 2B) in all CPGs, but differ from both bulk and surface water: with respect to surface water, the shape and the bandwidth of the transient absorption are different; yet, contrary to bulk water no excited-state absorption is observed. The latter observation is even more surprising considering that there is no difference between the steady-state absorption spectra of bulk and nanoconfined water (Figure 2A). To translate the data presented in Figure 2A and B into fundamental hydrogen-bond properties, we computed the OH frequency using a modified Lippincott–Schroeder (LS) potential function for the O H···O interactions. The absorption bands (static and transient) were calculated according to the procedure of Bakker et al. These calculations rationalize the observed differences in Figure 2B as an approximately 100 cm 1 red shift of the excited-state absorption band of confined water with respect to bulk water (Figure S6 in the Supporting Information). Therefore, one has to invoke a substantial confinement-induced enhancement of the anharmonic character of the OH vibrator. Figure 1. Anisotropy (r(t) on a logarithmic scale) decay of surface water in a low-hydration 1 nm porous glass (+) and water confined in fully hydrated 50 nm (~), 13 nm (*), and 1 nm (&) porous glasses (lines correspond to a single-exponential fit).

A Powerful Antiradiation Compound Revealed by a New High-Throughput Screening Method

We present a new high‐throughput screening method for the selection of powerful water‐soluble antiradiation compounds. This method, which uses conventional immunoassay techniques, allowed the capacity of a given compound to protect thymidine from irradiation to be evaluated. By applying this assay to an antioxidant library, we showed for the first time that norbadione A, a well‐known mushroom pigment, has pronounced atypical antiradiation properties.