Assessment and monitoring of water quality of the gulf of Morbihan, a littoral ecosystem under high anthropic pressure.

This field study is intended to propose a global methodology to assess and monitor the water quality of the gulf of Morbihan, a littoral ecosystem under increasing anthropic pressure. To this end, the Locmariaquer site, where Crassostrea gigas is extensively cultivated, was selected to perform a one-year follow-up of tissular glutathione S-transferase and acetylcholinesterase specific activities in this filter feeder organism. Calculation of an integrated index, corresponding to the ratio of the two enzymes activities, allowed to discriminate from the environmental noise, several clusters which could be representative environmental stress, potentially latent pollution. Moreover, the estrogenic activity was assessed in water samples collected at Locmariaquer and other strategic sites of the gulf. The results evidenced a low estrogenic-disrupting compound contamination of waters. Overall, this methodology produced an accurate outlook of a basal state for the gulf and could be developed in the context of a chronic monitoring of this site.

Interplay of flexibility and stability in the control of estrogen receptor activity.

Previously, we have identified an imperfect estrogen response element (rtERE) in the promoter of the rainbow trout vitellogenin gene. Although this ERE leads to a lower transcriptional activation, a better estradiol stimulation in vivo as compared to consensus ERE (EREcs) was observed. Here we examine the ability of recombinant human estrogen receptor alpha (rhERalpha) to bind DNA containing the EREcs or the natural imperfect rtERE, which contains three mismatches. At low salt concentration, whatever the ERE sequence, dissociation equilibrium constants of the specific rhERalpha-ERE complexes are similar (K(D) = 2 nM) with the same stoichiometry. As salt concentration increases from 80 to 200 mM KCl, the affinity of the rhERalpha-rtERE complex largely diminishes whereas that of rhERalpha-EREcs seems less affected. Hence the nature of the interactions stabilizing these complexes is different: more ionic in rhERalpha-rtERE as compared to rhERalpha-EREcs. Moreover, kinetic measurements showed that specific rhERalpha-ERE complexes exhibit shorter half-lives (few seconds) and that the rhERalpha-EREcs complex is more stable (33 s) than the complex that formed with rtERE (19.8 s), in accordance with equilibrium binding results. Finally, dynamic studies of rhERalpha have shown that the protein fluctuations are damped when the salt concentration increases or when bound to ERE and all the more with rtERE. The interplay of affinity, complex half-lives, and protein dynamics in the transcriptional regulation of estrogen receptor is discussed.

Myb-DNA recognition: role of tryptophan residues and structural changes of the minimal DNA binding domain of c-Myb.

The Myb oncoprotein specifically binds DNA by a domain composed of three imperfect repeats, R1, R2, and R3, each containing 3 tryptophans. The tryptophan fluorescence of the minimal binding domain, R2R3, of c-Myb was used to monitor structural flexibility changes occurring upon DNA binding to R2R3. The quenching of the Trp fluorescence by DNA titration shows that four out of the six tryptophans are involved in the formation of the specific R2R3-DNA complex and the environment of the tryptophan residues becomes more hydrophobic in the complex. The fluorescence intensity quenching of the tryptophans by binding of R2R3 to DNA is consistent with the decrease of the decay time: 1.46 ns for free R2R3 to 0.71 ns for the complexed protein. In the free R2R3, the six tryptophans are equally accessible to the iodide and acrylamide quenchers with a high collisional rate constant (4 x 10(9) and 3 x 10(9) M-1 s-1, respectively), indicating that R2R3 in solution is very flexible. In the R2R3-DNA complex, no Trp fluorescence quenching is observed with iodide whereas all tryptophan residues remain accessible to acrylamide with a collisional rate constant slightly slower than that in the free state. These results indicate that (i) a protein structural change occurs and (ii) the R2R3 molecule keeps a high mobility in the complex. The complex formation presents a two-step kinetics: a fast step corresponding to the R2R3-DNA association (7 x 10(5) M-1 s-1) and a slower one (0.004 s-1), which should correspond to a structural reorganization of the protein including a reordering of the water molecules at the protein-DNA interface.

An infrared study of 2H-bond variation in myoglobin revealed by high pressure.

A high-pressure Fourier-transform infrared technique was used to probe the evolution of 2H bonds inside the helical segments of myoglobin in relation to p2H, Tris concentration in the medium and iron-ligand nature. The analysis was focused on changes in the conformation-sensitive amide-I' band, reflecting the peptide C = O group stretching vibrations coupled to the in-plane N-2H bending and C = N stretching modes. From data obtained under high pressure, the strength of 2H bonds, inside the alpha-helical segments of the protein at atmospheric pressure, is not simply a function of p2H and salt concentration. At low Tris concentration (50 mM), the strength of these 2H bonds increases with p2H, whereas for a higher Tris concentration (100 mM) this strength is lower at p2H 7 than at p2H 6.0 or 8.5. It is also observed that the azidometmyoglobin molecule exhibits tighter intrahelical interactions and lower sensitivity to pressure than aquametmyoglobin. Information is also presented regarding interhelical interactions in relation to the solvent.

Variations of electrostatic interactions in myoglobin probed by UVRR spectroscopy. Effect of iron ligand and pH on tryptophan and tyrosine vibrations.

The present work investigates the variations of electrostatic interactions within the myoglobin molecule associated with azide heme binding and pH variations. Far ultraviolet (223 nm) resonance Raman spectroscopy of the tryptophan and tyrosine residues, along with acid-base titration measurements, have been used to monitor variations in the protein matrix. With previously determined mode assignments, it is shown that the Trp and Tyr residues of the globin moiety are influenced by the charge spatial distribution. Upon ligand binding or under various pH conditions, the polar interactions inside the protein appear to be modulated by the electric field generated by the charge array. It is concluded that the binding site properties of myoglobin can be modulated by the charge spatial distribution within the protein, even in the absence of measurable conformational changes of the bulk.

XANES spectroscopy sensitivity to small electronic changes. Case of carp azidomethemoglobin.

Spin states equilibrium of hemoglobin-iron varies with external conditions: pH, allosteric effectors, temperature. The small electronic reorganization of the iron caused by the spin state changes has been detected by X-ray absorption near edge structure (XANES) spectroscopy at room temperature. The iron K-edge region which is sensitive to spin state is located in 7110-7130 eV. Here are presented the 100% high spin and 100% low spin XANES spectra of carp azido ferric hemoglobin.