La sesta estinzione di massa

Mass extinction is when the referee blows the whistle: there is no objective definition of the term. Just as there is no precise discontinuity between background extinctions and mass extinctions, since there have been periods when biodiversity has declined more or less rapidly and profoundly (Wang 2003). However, it is accepted to identify five moments when there has been a particularly rapid and drastic decline in species diversity (Raup 1982), the last of which was the famous late Cretaceous extinction, 66 million years ago.Mass extinction is when the referee blows the whistle: there is no objective definition of the term. Just as there is no precise discontinuity between background extinctions and mass extinctions, since there have been periods when biodiversity has declined more or less rapidly and profoundly (Wang 2003). However, it is accepted to identify five moments when there has been a particularly rapid and drastic decline in species diversity (Raup 1982), the last of which was the famous late Cretaceous extinction, 66 million years ago.Mass extinction is when the referee blows the whistle: there is no objective definition of the term. Just as there is no precise discontinuity between background extinctions and mass extinctions, since there have been periods when biodiversity has declined more or less rapidly and profoundly (Wang 2003). However, it is accepted to identify five moments when there has been a particularly rapid and drastic decline in species diversity (Raup 1982), the last of which was the famous late Cretaceous extinction, 66 million years ago

Conformational equilibria in monomeric alpha-synuclein at the single molecule level

Natively unstructured proteins defy the classical "one sequence-one structure" paradigm of protein science. Monomers of these proteins in pathological conditions can aggregate in the cell, a process that underlies socially relevant neurodegenerative diseases such as Alzheimer and Parkinson. A full comprehension of the formation and structure of the so-called misfolded intermediates from which the aggregated states ensue is still lacking. We characterized the folding and the conformational diversity of alpha-synuclein (aSyn), a natively unstructured protein involved in Parkinson disease, by mechanically stretching single molecules of this protein and recording their mechanical properties. These experiments permitted us to directly observe directly and quantify three main classes of conformations that, under in vitro physiological conditions, exist simultaneously in the aSyn sample, including disordered and "beta-like" structures. We found that this class of "beta-like" structures is directly related to aSyn aggregation. In fact, their relative abundance increases drastically in three different conditions known to promote the formation of aSyn fibrils: the presence of Cu2+, the occurrence of the pathogenic A30P mutation, and high ionic strength. We expect that a critical concentration of aSyn with a "beta-like" structure must be reached to trigger fibril formation. This critical concentration is therefore controlled by a chemical equilibrium. Novel pharmacological strategies can now be tailored to act upstream, before the aggregation process ensues, by targeting this equilibrium. To this end, Single Molecule Force Spectroscopy can be an effective tool to tailor and test new pharmacological agents.Comment: 37 pages, 9 figures (including supplementary material

The Interplay between Chemistry and Mechanics in the Transduction of a Mechanical Signal into a Biochemical Function

There are many processes in biology in which mechanical forces are generated. Force-bearing networks can transduce locally developed mechanical signals very extensively over different parts of the cell or tissues. In this article we conduct an overview of this kind of mechanical transduction, focusing in particular on the multiple layers of complexity displayed by the mechanisms that control and trigger the conversion of a mechanical signal into a biochemical function. Single molecule methodologies, through their capability to introduce the force in studies of biological processes in which mechanical stresses are developed, are unveiling subtle intertwining mechanisms between chemistry and mechanics and in particular are revealing how chemistry can control mechanics. The possibility that chemistry interplays with mechanics should be always considered in biochemical studies.Comment: 50 pages, 18 figure

Finding common structural traits of GPCRs by computational molecular biology approaches

G-protein coupled receptors (GPCRs) are one of the most ancient, ubiquitous and functionally pervasive families of plasma membrane receptors. It is impossible to overstate their importance: The 800 GPCRs encoded in the human genome are involved in a staggering amount of physiological processes, from sensing to hormones to nervous signal transmission. At least 30% of currently marketed drugs target GPCRs. Despite recent technological advances, GPCR structural understanding is still in its infancy, with only 4% of unique receptors having a known structure. Our mechanistic understanding of GPCR function thus lags behind the demands of molecular biology, pharmacology and medicine. In this thesis, we used computational biology techniques to complement experimental biological information, tackling the general problem of the GPCR ligand recognition and signal transduction. The first study describes mechanistically the binding of the agonist strychnine to the TAS2R46 bitter taste receptor, a GPCR with no close structural template. By careful application of homology modeling and hybrid molecular mechanics/coarse grained molecular dynamics, guided by mutagenesis experiments, we obtained one of the most advanced structural studies on TAS2R46 to date. The work here described explaines mechanistically a significant amount of experimental data. It also suggests a new model for the remarkable agonist range of chemosensory receptors. The approach applied here is a successful benchmark for the possibility of using efficient, advanced molecular dynamics algorithms to in silico drug screening. In the second case, using structural bioinformatics tools for statistical analysis of crystal structures, validated by published mutagenesis information, we uncovered a novel switch involved in activation. We also confirmed the energetical conservation of several other switches, so far only proposed on the basis of a few structures, across all GPCRs structurally solved so far. The network we uncovered links the GPCR binding cavity to the intracellular space, and gives a quantitative foundation to the GPCRs activation models proposed in the literature. We also revealed an unexpected link between the activation mechanism and the receptor specificity in the cognate G-protein signalling pathway

Each one teaches one: characterizing active forms of proteins by single molecule force spectroscopy

This Ph.D. candidate thesis collects the research work I conducted under the supervision of Prof.Bruno Samor´ı in 2005,2006 and 2007. Some parts of this work included in the Part III have been begun by myself during my undergraduate thesis in the same laboratory and then completed during the initial part of my Ph.D. thesis: the whole results have been included for the sake of understanding and completeness. During my graduate studies I worked on two very different protein systems. The theorical trait d’union between these studies, at the biological level, is the acknowledgement that protein biophysical and structural studies must, in many cases, take into account the dynamical states of protein conformational equilibria and of local physico-chemical conditions where the system studied actually performs its function. This is introducted in the introductory part in Chapter 2. Two different examples of this are presented: the structural significance deriving from the action of mechanical forces in vivo (Chapter 3) and the complexity of conformational equilibria in intrinsically unstructured proteins and amyloid formation (Chapter 4). My experimental work investigated both these examples by using in both cases the single molecule force spectroscopy technique (described in Chapter 5 and Chapter 6). The work conducted on angiostatin focused on the characterization of the relationships between the mechanochemical properties and the mechanism of action of the angiostatin protein, and most importantly their intertwining with the further layer of complexity due to disulfide redox equilibria (Part III). These studies were accompanied concurrently by the elaboration of a theorical model for a novel signalling pathway that may be relevant in the extracellular space, detailed in Chapter 7.2. The work conducted on -synuclein (Part IV) instead brought a whole new twist to the single molecule force spectroscopy methodology, applying it as a structural technique to elucidate the conformational equilibria present in intrinsically unstructured proteins. These equilibria are of utmost interest from a biophysical point of view, but most importantly because of their direct relationship with amyloid aggregation and, consequently, the aetiology of relevant pathologies like Parkinson’s disease. The work characterized, for the first time, conformational equilibria in an intrinsically unstructured protein at the single molecule level and, again for the first time, identified a monomeric folded conformation that is correlated with conditions leading to -synuclein and, ultimately, Parkinson’s disease. Also, during the research work, I found myself in the need of a generalpurpose data analysis application for single molecule force spectroscopy data analysis that could solve some common logistic and data analysis problems that are common in this technique. I developed an application that addresses some of these problems, herein presented (Part V), and that aims to be publicly released soon

Inside the small length and energy scales of the world of the individual biological molecules.

Atomic force microscopy (AFM) has proved to be an essential tool of structural biology, being able not only to image but also to manipulate single biological molecules. These techniques make it possible to investigate the nanometer scale structure of single biological macromolecules and to study how an external force drives single biological molecules towards non-equilibrium conformations, by stretching and breaking bonds and interactions. This chapter focuses on the capabilities of the AFM-based single molecule methodologies to bring us into the nanometer-scale world of the single DNA molecules and into the pico-Newton force-scales of the interactions that sustain the protein folding

GOMoDo: A GPCRs Online Modeling and Docking Webserver

G-protein coupled receptors (GPCRs) are a superfamily of cell signaling membrane proteins that include >750 members in the human genome alone. They are the largest family of drug targets. The vast diversity and relevance of GPCRs contrasts with the paucity of structures available: only 21 unique GPCR structures have been experimentally determined as of the beginning of 2013. User-friendly modeling and small molecule docking tools are thus in great demand. While both GPCR structural predictions and docking servers exist separately, with GOMoDo (GPCR Online Modeling and Docking), we provide a web server to seamlessly model GPCR structures and dock ligands to the models in a single consistent pipeline. GOMoDo can automatically perform template choice, homology modeling and either blind or information-driven docking by combining together proven, state of the art bioinformatic tools. The web server gives the user the possibility of guiding the whole procedure. The GOMoDo server is freely accessible at http://molsim.sci.univr.it/gomodo

Structure/Function Relationships of Phospholipases C Beta

Phospholipases C beta (PLC-βs) are essential components of the signal transduction of metazoans. They catalyze the production of the second messengers inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) from the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2). These enzymes are activated by G-protein-coupled receptors (GPCRs) through the interaction with the alpha subunit of heterotrimeric G-proteins belonging to the Gq family (Gαq), the Gβγ subunits released by the inhibitory G-protein (Gi) and Ca2+ ions. Here we review current structural insights on these important proteins, with a particular focus on the most structurally characterized isoform (PLC-β3) and the activation mechanism operated by Gαq. We propose, following the lead of recent studies, that a tight combination of experiments and molecular simulations are instrumental in further enlightening the structure/function understanding of PLC-βs. - See more at: http://www.eurekaselect.com/116059/article#sthash.cojH1BB1.dpu

Evidence for a Transient Additional Ligand Binding Site in the TAS2R46 Bitter Taste Receptor

Most human G protein coupled receptors (GPCRs) are activated by small molecules binding to their 7-transmembrane (7-TM) helix bundle. They belong to basally diverging branches: the 25 bitter taste 2 receptors and most members of the very large rhodopsin-like/class A GPCRs subfamily. Some members of the class A branch have been suggested to feature not only an orthosteric agonist-binding site but also a more extracellular or “vestibular” site, involved in the binding process. Here we use a hybrid molecular mechanics/coarse-grained (MM/CG) molecular dynamics approach on a widely studied bitter taste receptor (TAS2R46) receptor in complex with its agonist strychnine. Three ∼1 μs molecular simulation trajectories find two sites hosting the agonist, which together elucidate experimental data measured previously and in this work. This mechanism shares similarities with the one suggested for the evolutionarily distant class A GPCRs. It might be instrumental for the remarkably broad but specific spectrum of agonists of these chemosensory receptors