Abstract
Over the last decades, a wide range of biophysical techniques investigating protein-ligand interactions have become indispensable tools to complement high-resolution crystal structure determinations. Current approaches in solution range from high-throughput-capable methods such as thermal shift assays (TSA) to highly accurate techniques including microscale thermophoresis (MST) and isothermal titration calorimetry (ITC) that can provide a full thermodynamic description of binding events. Surface-based methods such as surface plasmon resonance (SPR) and dual polarization interferometry (DPI) allow real-time measurements and can provide kinetic parameters as well as binding constants. DPI provides additional spatial information about the binding event. Here, an account is presented of new developments and recent applications of TSA and DPI connected to crystallography.
Highlights
The biophysical characterization of protein–ligand relations ranging from protein–ion and protein–drug interactions to protein–protein and protein–nucleic acid interactions plays a key role in structural biology
Experimental results of dual polarization interferometry (DPI) measurements with human serum albumin (HSA) immobilized on the sensor chip
Thermal shift assays can be established and performed using standard equipment that is present in almost every molecular-biology or macromolecular laboratory
Summary
The biophysical characterization of protein–ligand relations ranging from protein–ion and protein–drug interactions to protein–protein and protein–nucleic acid interactions plays a key role in structural biology. Methods routinely used to screen compound libraries span from highly specialized techniques requiring significant instrumentation and expertise such as NMR (Sillerud & Larson, 2012) and mass spectrometry (Hofstadler & Sannes-Lowery, 2006) to simpler methods including thermal shift assays (TSA) that determine a shift in melting temperature typically measured by changes in light scattering or by fluorescence techniques. A wide range of more sophisticated biophysical techniques can be used to further characterize biomolecular interactions. Surface plasmon resonance (SPR) biosensor techniques available from various companies are used to determine kinetic parameters as well as the binding constants (typically in the range from subnanomolar to low millimolar) and stoichiometries of biomolecular interactions. SPR measurements are normally performed with intermediate throughput and are rarely applicable to chemical library screening.
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