1. Saturation and Competitive Binding Expriments Anton Zavialov Uppsala

2. Binding is an essential functional step of many processes

3. Protein-ligand interactions Hydrophobic effect Van der Waals interactions Hydrogen bonds Salt bridges

4. Hydrophobic effect

5. Saturation Binding Experiment Main Parameters: 1.Affinity: dissociation constant (Kd)  Gd o =- RT ln(Kd) 2. Total amount of receptors (R T ) or stoichiometry of the binding (n, m) Additional information: Type of the binding (cooperative or non- cooperative), site accessibility, site heterogeneity, etc.

6. Saturation Curve and Scatchard Plot [RL]=R T [L] / (Kd+[L])[RL] / [L] =R T / Kd - [RL] /Kd

7. Effects

8. Experimental setup

10. Technical considerations 1.R T <<L T, which means that [L] is close to L T -life is easier, but to create high concentration of the ligand we have to add non-radioactive ”cold” ligand 2. Which range of concentrations to use? - around Kd and above: e.g. expected Kd=2-4  M, use L T =2-30  M Protein concentration? About 0.2  M in this case

11. My Last Weekend’s Binding Experiment

12. Competitive Binding Experiment Advantage: Cheep Ligand is labeled, whereas inhibitors are not – I1, I2, I3, I4...

13. My Last Weekend’s Binding Experiment [RL]i/[RL]=1/(1+[I]/IC50), Ki=IC50/(1+L T /K l ), but only when R T <<L T and R T <<I T