G-protein coupled receptors (GPCRs) are a superfamily of cell signaling membrane proteins that in... more 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
Hooke is an open source, extensible software intended for analysis of atomic force microscope (AF... more Hooke is an open source, extensible software intended for analysis of atomic force microscope (AFM)-based single molecule force spectroscopy (SMFS) data. We propose it as a platform on which published and new algorithms for SMFS analysis can be integrated in a standard, open fashion, as a general solution to the current lack of a standard software for SMFS data analysis. Specific features and support for file formats are coded as independent plugins. Any user can code new plugins, extending the software capabilities. Basic automated dataset filtering and semi-automatic analysis facilities are included. Availability: Software and documentation are available at
Here, we use single-molecule techniques to study the aggregation of a-synuclein, the protein whos... more Here, we use single-molecule techniques to study the aggregation of a-synuclein, the protein whose misfolding and deposition is associated with Parkinson's disease. We identify a conformational change from the initially formed oligomers to stable, more compact proteinase-K-resistant oligomers as the key step that leads ultimately to fibril formation. The oligomers formed as a result of the structural conversion generate much higher levels of oxidative stress in rat primary neurons than do the oligomers formed initially, showing that they are more damaging to cells. The structural conversion is remarkably slow, indicating a high kinetic barrier for the conversion and suggesting that there is a significant period of time for the cellular protective machinery to operate and potentially for therapeutic intervention, prior to the onset of cellular damage. In the absence of added soluble protein, the assembly process is reversed and fibrils disaggregate to form stable oligomers, hence acting as a source of cytotoxic species.
By studying with the single molecule force spectroscopy (SMFS) methodology the mechanical behavio... more By studying with the single molecule force spectroscopy (SMFS) methodology the mechanical behaviour of single biomolecules, we are learning how mechanical forces like those present in the extracellular space modify the conformation of proteins, possibly leading to functional switches. We also understand that the functional efficiency of those mechanical switches can rely on their coupling to some independent biochemical control of the protein conformational changes. The disulfide bonds have been recently proposed to act as potential redox switches, even if their structural bases are unclear. Here we discuss, also on the basis of experimental evidences based on SMFS, the possibility that disulfide bond switching and mechanical deformation of extracellular proteins can be coupled, thus leading to an efficient and highly tuned switch for protein function. We propose this as one of the biological mechanisms that regulate extracellular protein functionality.
Bioinformatics/computer Applications in The Biosciences, 2009
Hooke is an open source, extensible software intended for analysis of atomic force microscope (AF... more Hooke is an open source, extensible software intended for analysis of atomic force microscope (AFM)-based single molecule force spectroscopy (SMFS) data. We propose it as a platform on which published and new algorithms for SMFS analysis can be integrated in a standard, open fashion, as a general solution to the current lack of a standard software for SMFS data analysis. Specific features and support for file formats are coded as independent plugins. Any user can code new plugins, extending the software capabilities. Basic automated dataset filtering and semi-automatic analysis facilities are included. Availability: Software and documentation are available at
Natively unstructured proteins defy the classical "one sequence-one structure" paradigm of protei... more 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.
There are many processes in biology in which mechanical forces are generated. Force-bearing netwo... more 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.
Inside the Small Length and Energy Scales of the World of the Individual Biological Molecule
Atomic force microscopy (AFM) has proved to be an essential tool of structural biology, being abl... more 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 nonequilibrium 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.
Over the past two decades, remarkable developments and refinements in single-molecule force spect... more Over the past two decades, remarkable developments and refinements in single-molecule force spectroscopy (SMFS) techniques have been achieved, thus providing new investigation tools for biophysical and biological research. The potential of these techniques is demonstrated by the rapidly growing number of applications and reports, and by the fact that these once cutting-edge techniques are currently becoming more reliable and precise. In a classical SMFS experiment , the cantilever tip is pressed onto a layer of proteins, immobilized on a substrate, followed by the extension of the protein molecules through the retraction of the piezoelectric positioner. Such an approach allows the measurement of the forces required to unfold protein domains, with piconewton sensitivity, and of the consequent changes of the protein length, with angstrom resolution. The technique acts directly on the protein molecule (that is maintained under physiological conditions), leaving its environment unchanged. SMFS is capable to monitor a variety of interactions between the stylus tip and the sample. However, upon interaction of the cantilever tip with the protein layer and the subsequent withdrawal of the piezoelectric positioner, many protein molecules interacting with the tip exert a force that bends the cantilever. Hence, the early part of the force-extension curve is frequently characterized by large fluctuations in force. This typically poses two difficulties in the interpretation of force-extension curves. First, the forces involved in the extension of short molecules may be masked by the noise caused by such nonspecific interactions. Second, almost all experiments show force peaks reflecting such nonspecific interactions, so that a means is required to differentiate true unfolding events from spurious interactions. The solution to this problem was found in molecular biology. By ligating multiple copies of the cDNA encoding a specific Figure 1. Schematic representation of a typical SMFS experiment on a polyprotein. A) The tip of the cantilever is brought into contact with proteins covalently immobilized onto a substrate, followed by the retraction of the piezoelectric positioner. The interaction of the tip with the proteins results in the bending of the cantilever. B) The stretching of the reference modules (such as I27) produces force-extension curves with a characteristic saw-tooth pattern. The individual saw-tooth peaks correspond to the sequential unraveling of individual domains in the reference multimodular protein. The unfolding force is a measure of the mechanical stability of the protein domains.
G-protein coupled receptors (GPCRs) are a superfamily of cell signaling membrane proteins that in... more 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
Hooke is an open source, extensible software intended for analysis of atomic force microscope (AF... more Hooke is an open source, extensible software intended for analysis of atomic force microscope (AFM)-based single molecule force spectroscopy (SMFS) data. We propose it as a platform on which published and new algorithms for SMFS analysis can be integrated in a standard, open fashion, as a general solution to the current lack of a standard software for SMFS data analysis. Specific features and support for file formats are coded as independent plugins. Any user can code new plugins, extending the software capabilities. Basic automated dataset filtering and semi-automatic analysis facilities are included. Availability: Software and documentation are available at
Here, we use single-molecule techniques to study the aggregation of a-synuclein, the protein whos... more Here, we use single-molecule techniques to study the aggregation of a-synuclein, the protein whose misfolding and deposition is associated with Parkinson's disease. We identify a conformational change from the initially formed oligomers to stable, more compact proteinase-K-resistant oligomers as the key step that leads ultimately to fibril formation. The oligomers formed as a result of the structural conversion generate much higher levels of oxidative stress in rat primary neurons than do the oligomers formed initially, showing that they are more damaging to cells. The structural conversion is remarkably slow, indicating a high kinetic barrier for the conversion and suggesting that there is a significant period of time for the cellular protective machinery to operate and potentially for therapeutic intervention, prior to the onset of cellular damage. In the absence of added soluble protein, the assembly process is reversed and fibrils disaggregate to form stable oligomers, hence acting as a source of cytotoxic species.
By studying with the single molecule force spectroscopy (SMFS) methodology the mechanical behavio... more By studying with the single molecule force spectroscopy (SMFS) methodology the mechanical behaviour of single biomolecules, we are learning how mechanical forces like those present in the extracellular space modify the conformation of proteins, possibly leading to functional switches. We also understand that the functional efficiency of those mechanical switches can rely on their coupling to some independent biochemical control of the protein conformational changes. The disulfide bonds have been recently proposed to act as potential redox switches, even if their structural bases are unclear. Here we discuss, also on the basis of experimental evidences based on SMFS, the possibility that disulfide bond switching and mechanical deformation of extracellular proteins can be coupled, thus leading to an efficient and highly tuned switch for protein function. We propose this as one of the biological mechanisms that regulate extracellular protein functionality.
Bioinformatics/computer Applications in The Biosciences, 2009
Hooke is an open source, extensible software intended for analysis of atomic force microscope (AF... more Hooke is an open source, extensible software intended for analysis of atomic force microscope (AFM)-based single molecule force spectroscopy (SMFS) data. We propose it as a platform on which published and new algorithms for SMFS analysis can be integrated in a standard, open fashion, as a general solution to the current lack of a standard software for SMFS data analysis. Specific features and support for file formats are coded as independent plugins. Any user can code new plugins, extending the software capabilities. Basic automated dataset filtering and semi-automatic analysis facilities are included. Availability: Software and documentation are available at
Natively unstructured proteins defy the classical "one sequence-one structure" paradigm of protei... more 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.
There are many processes in biology in which mechanical forces are generated. Force-bearing netwo... more 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.
Inside the Small Length and Energy Scales of the World of the Individual Biological Molecule
Atomic force microscopy (AFM) has proved to be an essential tool of structural biology, being abl... more 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 nonequilibrium 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.
Over the past two decades, remarkable developments and refinements in single-molecule force spect... more Over the past two decades, remarkable developments and refinements in single-molecule force spectroscopy (SMFS) techniques have been achieved, thus providing new investigation tools for biophysical and biological research. The potential of these techniques is demonstrated by the rapidly growing number of applications and reports, and by the fact that these once cutting-edge techniques are currently becoming more reliable and precise. In a classical SMFS experiment , the cantilever tip is pressed onto a layer of proteins, immobilized on a substrate, followed by the extension of the protein molecules through the retraction of the piezoelectric positioner. Such an approach allows the measurement of the forces required to unfold protein domains, with piconewton sensitivity, and of the consequent changes of the protein length, with angstrom resolution. The technique acts directly on the protein molecule (that is maintained under physiological conditions), leaving its environment unchanged. SMFS is capable to monitor a variety of interactions between the stylus tip and the sample. However, upon interaction of the cantilever tip with the protein layer and the subsequent withdrawal of the piezoelectric positioner, many protein molecules interacting with the tip exert a force that bends the cantilever. Hence, the early part of the force-extension curve is frequently characterized by large fluctuations in force. This typically poses two difficulties in the interpretation of force-extension curves. First, the forces involved in the extension of short molecules may be masked by the noise caused by such nonspecific interactions. Second, almost all experiments show force peaks reflecting such nonspecific interactions, so that a means is required to differentiate true unfolding events from spurious interactions. The solution to this problem was found in molecular biology. By ligating multiple copies of the cDNA encoding a specific Figure 1. Schematic representation of a typical SMFS experiment on a polyprotein. A) The tip of the cantilever is brought into contact with proteins covalently immobilized onto a substrate, followed by the retraction of the piezoelectric positioner. The interaction of the tip with the proteins results in the bending of the cantilever. B) The stretching of the reference modules (such as I27) produces force-extension curves with a characteristic saw-tooth pattern. The individual saw-tooth peaks correspond to the sequential unraveling of individual domains in the reference multimodular protein. The unfolding force is a measure of the mechanical stability of the protein domains.
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Papers by Massimo Sandal