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      Protein-binding assays in biological liquids using microscale thermophoresis

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          Abstract

          Protein interactions inside the human body are expected to differ from the situation in vitro. This is crucial when investigating protein functions or developing new drugs. In this study, we present a sample-efficient, free-solution method, termed microscale thermophoresis, that is capable of analysing interactions of proteins or small molecules in biological liquids such as blood serum or cell lysate. The technique is based on the thermophoresis of molecules, which provides information about molecule size, charge and hydration shell. We validated the method using immunologically relevant systems including human interferon gamma and the interaction of calmodulin with calcium. The affinity of the small-molecule inhibitor quercetin to its kinase PKA was determined in buffer and human serum, revealing a 400-fold reduced affinity in serum. This information about the influence of the biological matrix may allow to make more reliable conclusions on protein functionality, and may facilitate more efficient drug development.

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          Most cited references31

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          Why molecules move along a temperature gradient.

          Molecules drift along temperature gradients, an effect called thermophoresis, the Soret effect, or thermodiffusion. In liquids, its theoretical foundation is the subject of a long-standing debate. By using an all-optical microfluidic fluorescence method, we present experimental results for DNA and polystyrene beads over a large range of particle sizes, salt concentrations, and temperatures. The data support a unifying theory based on solvation entropy. Stated in simple terms, the Soret coefficient is given by the negative solvation entropy, divided by kT. The theory predicts the thermodiffusion of polystyrene beads and DNA without any free parameters. We assume a local thermodynamic equilibrium of the solvent molecules around the molecule. This assumption is fulfilled for moderate temperature gradients below a fluctuation criterion. For both DNA and polystyrene beads, thermophoretic motion changes sign at lower temperatures. This thermophilicity toward lower temperatures is attributed to an increasing positive entropy of hydration, whereas the generally dominating thermophobicity is explained by the negative entropy of ionic shielding. The understanding of thermodiffusion sets the stage for detailed probing of solvation properties of colloids and biomolecules. For example, we successfully determine the effective charge of DNA and beads over a size range that is not accessible with electrophoresis.
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            Structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine.

            The specific phosphoinositide 3-kinase (PI3K) inhibitors wortmannin and LY294002 have been invaluable tools for elucidating the roles of these enzymes in signal transduction pathways. The X-ray crystallographic structures of PI3Kgamma bound to these lipid kinase inhibitors and to the broad-spectrum protein kinase inhibitors quercetin, myricetin, and staurosporine reveal how these compounds fit into the ATP binding pocket. With a nanomolar IC50, wortmannin most closely fits and fills the active site and induces a conformational change in the catalytic domain. Surprisingly, LY294002 and the lead compound on which it was designed, quercetin, as well as the closely related flavonoid myricetin bind PI3K in remarkably different orientations that are related to each other by 180 degrees rotations. Staurosporine/PI3K interactions are reminiscent of low-affinity protein kinase/staurosporine complexes. These results provide a rich basis for development of isoform-specific PI3K inhibitors with therapeutic potential.
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              Direct measurement of protein binding energetics by isothermal titration calorimetry.

              Of all the techniques that are currently available to measure binding, isothermal titration calorimetry is the only one capable of measuring not only the magnitude of the binding affinity but also the magnitude of the two thermodynamic terms that define the binding affinity: the enthalpy (AH) and entropy (AS) changes. Recent advances in instrumentation have facilitated the development of experimental designs that permit the direct measurement of arbitrarily high binding affinities, the coupling of binding to protonation/deprotonation processes and the analysis of binding thermodynamics in terms of structural parameters. Because isothermal titration calorimetry has the capability to measure different energetic contributions to the binding affinity, it provides a unique bridge between computational and experimental analysis. As such, it is increasingly becoming an essential tool in molecular design.
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                Author and article information

                Journal
                Nature Communications
                Nat Commun
                Springer Science and Business Media LLC
                2041-1723
                December 2010
                October 19 2010
                December 2010
                : 1
                : 1
                Article
                10.1038/ncomms1093
                20981028
                e80f21fb-e8e5-40c3-ac63-abde9d616d49
                © 2010

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