Serge Pin

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.

Antioxidant and Free Radical Scavenging Properties of Marennine, a Blue-Green Polyphenolic Pigment from the Diatom Haslea ostrearia (Gaillon/Bory) Simonsen Responsible for the Natural Greening of Cultured Oysters

Among microalgae, the marine diatom Haslea ostrearia has the distinctive feature of synthesizing and releasing, into the surrounding environment, a blue-green polyphenolic pigment called marennine. The oyster-breeding industry commonly makes use of this natural phenomenon for the greening of oysters grown in the ponds of the French Atlantic coast. This article reports the in vitro antioxidant properties of pure marennine. Two kinds of evaluation systems were adopted to test the antioxidative activity of marennine: antioxidant capacity assays (beta-carotene and thymidine protection assays and iron reducing power assay) and free radical scavenging assays (DPPH*, O2*-, and HO*). In almost all cases, marennine exhibited significantly higher antioxidative and free radical scavenging activities than natural and synthetic antioxidants commonly used in food, as shown by comparing median effective concentration (EC 50) values, for each test independently. This medium molecular weight polyphenol (around 10 kDa) from microalgae is thus a potentially useful natural antioxidant. Because of its blue-coloring property and water solubility, it could also be used as a natural food-coloring additive.

Förster energy transfer from tryptophan to flavin in glucose oxidase enzyme

Abstract The difference in the properties of the tryptophan fluorescence of the hologlucose oxidase forms, as compared to those of the apoprotein, can unambiguously be interpreted as due to a Forster energy transfer from the tryptophan residues to the flavinic group. Indeed, the atomic absorption of a glucose oxidase solution shows that the enzyme is not associated to any metals which could be responsible for the tryptophan fluorescence quenching effect. The data show that only 7 Trp residues are partially coupled to the flavin group and that the strength of this coupling is dependent on the flavin redox state.

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.

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.

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.

[13] Stationary and time-resolved circular dichroism of hemoglobins

Publisher Summary This chapter reviews the applications of circular dichroism (CD) spectroscopy to hemoglobin (Hb). This chapter discusses essentials of CD theory. Because many electronic states are involved in proteins, it is possible to investigate the molecule in different wavelength regions. Each region contains some information concerning a part of the three-dimensional (3D) organization of the macromolecule. By choosing a set of different wavelengths, different chromophores can be observed. In the spectral region below 250 nm, the optical activity is dominated by the peptide backbone organization. Protein structural analysis is interpreted by the existence of a correlation between CD spectra and the organization of the secondary structure: α helix, β sheet, and random coil. The CD spectrum of each secondary structure is assumed to be independent of others, and the resulting CD spectrum is a simple summation. In the chapter, two kinds of reference were used to analyze protein structure by CD.

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.

Synthesis and antioxidant properties of pulvinic acids analogues

The synthesis of three types of pulvinic acid analogues, using a diversity-oriented strategy starting from a single compound, dimethyl l-tartrate, is described. Lacey-Dieckmann condensation, alcohol dehydration and Suzuki-Miyaura cross-couplings were employed in the course of the analogues syntheses. The evaluation of the antioxidant properties of the 28 synthesized analogues was carried out using antioxidant capacity assays (protection of thymidine and β-carotene) and free radical scavenging assays (DPPH radical and ABTS radical cation). This allowed to assess the relative influence of the groups bonded to the tetronic ring and to the exocyclic double bond on the activity, as well as the importance of this exocyclic double bond. It was shown that the presence of an electron-donating group on the 3-position of the tetronic ring had a beneficial effect. It was shown in several assays that the presence of the exocyclic bond was not crucial to the activity.