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

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. MOLECULAR RECOGNITION
Essential for all physiological and pathological processes.
• Cell grouth and development
• Cell-cell communication
• Bacterial/viral infections
• Tumor development
• etc…..

3. MOLECULAR RECOGNITION
Ligand – receptor interactions
• Protein – protein
• Nucleic acid – protein
• Small molecule – biomolecule…….
Free species Complex
A + M AM

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. 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. ANALYSIS
Thermodynamics
Kinetics
Structure
Data
(graph(s))
How to get the information?
Monitor the interaction
(Biophysical technique)
Ligand – receptor interaction

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. 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. Thermodynamics
Raw Data
Dataseries
peak integration
injection peak

10. ANALYSIS
Thermodynamics
Kinetics
Structure
Data
(graph(s))
We can get much more!
Ligand – receptor interaction
ITC
Isothermal Titration Calorimetry (ITC)
ITC

11. Obtaining kinetic information from ITC
KinITC: obtaining kinetic information from ITC

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. AFFINImeter & KinITC
Analytical tools to study complex data
heat vs time (kinetic information!)
KA = kon/koff kon = KA·koff

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. Wieseman parameter
Stoichiometric ratio for current injection
KinITC: obtaining kinetic information from ITC
Equilibration time
Dissociation rate constant
AFFINImeter & KinITC

16. ANALYSIS
Thermodynamics
Kinetics
Structure
Data
(graph(s))
We can get much more!
Ligand – receptor interaction
ITC
Isothermal Titration Calorimetry (ITC)

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. Tailored binding model
Global fitting
ROBUST ANALYSIS
• Structural information.
• Binding mechanism.
• Cooperativiy.
• Competitive experiments.
MODEL BUILDER
AFFINImeter advanced tools

19. AFFINImeter advanced tools
Based on Site constants
Based on Stoichiometric constants
CaMBD
CaM
k1
k2
k2a
k1a
k1 k2a

20. Analytical tools to study complex ITC data