An Integrated Approach for Evaluating the Restoration of the Salinity Gradient in Transitional Waters: Monitoring and Numerical Modeling in the Life Lagoon Refresh Case Study

Large lagoons usually show a salinity gradient due to fresh water tributaries with inner areas characterized by lower mean values and higher fluctuation of salinity than seawaterdominated areas. In the Venice Lagoon, this ecotonal environment, characterized in the past by oligo‐mesohaline waters and large intertidal areas vegetated by reedbeds, was greatly reduced by historical human environmental modifications, including the diversion of main rivers outside the Venice Lagoon. The reduction of the fresh water inputs caused a marinization of the lagoon, with an increase in salinity and the loss of the related habitats, biodiversity, and ecosystem services. To counteract this issue, conservation actions, such as the construction of hydraulic infrastructures for the introduction and the regulation of a fresh water flow, can be implemented. The effectiveness of these actions can be preliminarily investigated and then verified through the combined implementation of environmental monitoring and numerical modeling. Through the results of the monitoring activity carried out in Venice Lagoon in the framework of the Life Lagoon Refresh (LIFE16NAT/IT/000663) project, the study of salinity is shown to be a successful and robust combination of different types of monitoring techniques. In particular, the characterization of salinity is obtained by the acquisition of continuous data, field campaigns, and numerical modeling

Structural characterization of a high affinity mononuclear site in the Copper(II)-alpha-Synuclein complex.

Human alpha-Synuclein (aS), a 140 amino acid protein, is the main constituent of Lewy bodies, the cytoplasmatic deposits found in the brains of Parkinson's disease patients, where it is present in an aggregated, fibrillar form. Recent studies have shown that aS is a metal binding protein. Moreover, heavy metal ions, in particular divalent copper, accelerate the aggregation process of the protein. In this work, we investigated the high affinity binding mode of truncated aS (1-99) (aS99) with Cu(II), in a stoichiometric ratio, to elucidate the residues involved in the binding site and the role of copper ions in the protein oligomerization. We used Electron Paramagnetic Resonance spectroscopy on the Cu(II)-aS99 complex at pH 6.5, performing both multifrequency continuous wave experiments and pulsed experiments at X-band. The comparison of 9.5 and 95 GHz data showed that at this pH only one binding mode is present. To identify the nature of the ligands, we performed Electron Spin Echo Envelope Modulation, Hyperfine Sublevel Correlation Spectroscopy, and pulsed Davies Electron-Nuclear Double Resonance (Davies-ENDOR) experiments. We determined that the EPR parameters are typical of a type-II copper complex, in a slightly distorted square planar geometry. Combining the results from the different pulsed techniques, we obtained that the equatorial coordination is {N(im), N(-), H(2)O, O}, where N(im) is the imino nitrogen of His50, N(-) a deprotonated amido backbone nitrogen that we attribute to His50, H(2)O an exchangeable water molecule, and O an unidentified oxygen ligand. Moreover, we propose that the free amino terminus (Met1) participates in the complex as an axial ligand. The MXAN analysis of the XAS k-edge absorption data allowed us to independently validate the structural features proposed on the basis of the magnetic parameters of the Cu(II)-aS99 complex and then to further refine the quality of the proposed structural model

Broken helix in vesicle and micelle-bound alpha-synuclein: Insights from site-directed spin labeling-EPR experiments and MD simulations

Human a-Synuclein (aS) is a 140 amino acid-protein strongly associated to both familial and sporadic cases of Parkinson\u2019s disease. The physiological role of aS is still elusive, although its nerve terminal localization and its ability to interact with membranes seem to be the key factors for its physiological functions. aS is a natively unfolded, soluble protein whose primary sequence is characterized by the presence of seven imperfect 11-residue repeats potentially able to fold into an amphipathic helix. In the presence of sodium dodecyl sulfate (SDS) micelles or synthetic membranes, the first ~100 residues of aS undergo a conformational transition to a helical state. We report here on an SDSL-EPR study of the controversial interhelix region of aS, when bound to either SUV or SDS micelles, accompanied by modeling of the spin label in the two proposed protein conformations. The investigation is completed by a Molecular Dynamics (MD) simulation of the 31-52 fragment interacting with a lipid bilayer. Our data show that the 38-44 region of aS exhibits a very similar behavior in micelles and in SUV. Specifically, we find evidence for a high degree of conformational disorder rather than for the formation of a continuous helical structure. The main result of our investigation is that SUV-bound aS bears most of the features reported for it in micellar environment: an unbroken helical structure of the region around residue 40 can be ruled out. Helix breaking does not appear to be a mere consequence of the constraints imposed by the small micellar dimensions, but as an intrinsic feature of aS, when bound to amphipathic interfaces. Furthermore, we can confirm the picture of the interhelix region as characterized by conformational disorder, rather than exhibiting a single structure. This disorder might play a role in aS binding to synaptic vesicles, by allowing the protein to fit into amphipathic aggregates with different degrees of lipid packing strain