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Affinimeter Tutorial

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AFFINImeter: una herramienta avanzadda para el análisis de datos de Calorimetría de Titulación Isotérmica (ITC)

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Affinimeter Tutorial

  1. 1. AFFINImeter: una herramienta avanzada para el análisis de datos de Calorimetría de Titulación Isotérmica (ITC) TUTORIAL 15 y 16 de Octubre de 2015
  2. 2. MOLECULAR RECOGNITION Essential for all physiological and pathological processes. • Cell grouth and development • Cell-cell communication • Bacterial/viral infections • Tumor development • etc…..
  3. 3. MOLECULAR RECOGNITION Ligand – receptor interactions • Protein – protein • Nucleic acid – protein • Small molecule – biomolecule……. Free species Complex A + M AM
  4. 4. o Understanding Biological proceses. o Pathological proceses. o Drug Discovery and development. Ligand – receptor interactions: characterization • Thermodynamics • Kinetics • Structure • How strong? • How fast? • Where/how?
  5. 5. Ligand – receptor interactions: characterization • Thermodynamics: heat, energy DG: free energy (K, binding constant) DH: enthalpy DS: entropy • Structure & mechanism: stoichiometry • Kinetics: speed kon: association rate constant koff: dissociation rate constant DG = RTLnKA DG = DH – TDS KA = kon/koff
  6. 6. ANALYSIS Thermodynamics Kinetics Structure Data (graph(s)) How to get the information? Monitor the interaction (Biophysical technique) Ligand – receptor interaction
  7. 7. ANALYSIS Thermodynamics Kinetics Structure Data (graph(s)) How to get the information? Ligand – receptor interaction Monitor the interaction (Biophysical technique) ITC Isothermal Titration Calorimetry (ITC)
  8. 8. Thermodynamics • Gold-standard technique for the thermodynamic characterization of interactions • Measures the heat change occured in the calorimetric cell throughout the titration ligand receptor The ITC titration: Power vs. Time plot (thermogram) ITC isotherm peak integration KA , DH, stoichiometry DG, DS
  9. 9. Thermodynamics Raw Data Dataseries peak integration injection peak
  10. 10. ANALYSIS Thermodynamics Kinetics Structure Data (graph(s)) We can get much more! Ligand – receptor interaction ITC Isothermal Titration Calorimetry (ITC) ITC
  11. 11. Obtaining kinetic information from ITC KinITC: obtaining kinetic information from ITC
  12. 12. Obtaining kinetic information from ITC • IS THERE KINETIC INFORMATION IN MY THERMOGRAM?: A VISUAL INSPECTION Mid titration: Peak broadening KinITC: obtaining kinetic information from ITC
  13. 13. AFFINImeter & KinITC Analytical tools to study complex data heat vs time (kinetic information!) KA = kon/koff kon = KA·koff
  14. 14. “Essentially, KinITC is based upon linking the kinetics of the reaction to heat power production in the measurement cell” = = = = concentration of titrand in the cell 1:1 binding Heat power KinITC: obtaining kinetic information from ITC AFFINImeter & KinITC
  15. 15. Wieseman parameter Stoichiometric ratio for current injection KinITC: obtaining kinetic information from ITC Equilibration time Dissociation rate constant AFFINImeter & KinITC
  16. 16. ANALYSIS Thermodynamics Kinetics Structure Data (graph(s)) We can get much more! Ligand – receptor interaction ITC Isothermal Titration Calorimetry (ITC)
  17. 17. Structure & Mechanism Simple 1:1 interaction • Competitive experiments. • Species dimerization. • Multi-site binding • Cooperative effects • etc Complex data, usually explained by the presence of several equilibria
  18. 18. Tailored binding model Global fitting ROBUST ANALYSIS • Structural information. • Binding mechanism. • Cooperativiy. • Competitive experiments. MODEL BUILDER AFFINImeter advanced tools
  19. 19. AFFINImeter advanced tools Based on Site constants Based on Stoichiometric constants CaMBD CaM k1 k2 k2a k1a k1 k2a
  20. 20. Analytical tools to study complex ITC data

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