Microcalorimetry as a tool for structural biology
Learn how Isothermal titration calorimetry can be used to guide protein (RNA)/ligand crystallization and to resolve common issues with crystallization of complexes.
Microcalorimetry as a Tool for Structural Biology
Learn how isothermal titration calorimetry can be used to guide protein (RNA)/ligand crystallization and to resolve common issues with crystallization of complexes.
Crystallization of complexes (protein/protein, protein/nucleic acid, protein/ligand, nucleic acids/nucleic acid, nucleic acid/ligand), even on well-characterized biological systems, are frequently tedious and either time or sample consuming. The success rate of complex crystallization can be significantly improved if a proper preliminary characterization of the complex using biophysical methods is performed. It well established that Dynamic Light Scattering (DLS) is key to evaluate sample crystallizability (1). Likewise, DSC thermofluor-based optimization strategy have been developed to facilitate protein/ligand crystallization (2). Isothermal Titration Calorimetry (ITC) is the “gold standard” technique for investigating molecular interactions and we show here that it can be a valuable technique to improve crystallization of complexes. ITC is a true in-solution technique that directly provides, in one single experiment, the complete binding profile between two molecules: binding affinity (Ka), enthalpy and entropy changes (ΔH and ΔS) and stoichiometry (N) between two molecules are obtained very accurately (3-5). A major advantage of ITC over other similar biophysical approaches is that it is not restricted by macromolecule upper or lower size limit, there is no buffer restriction, and, most importantly for structural studies, it does not required any labeling. In addition, we have recently shown that modern ITC apparatus and new processing methods also allow obtaining a complete kinetic description on more diverse systems than usually thought, ranging from simple ligand binding to complex RNA folding (6).
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