Saturation and Competitive Binding Expriments

Anton Zavialov

Uppsala

Binding is an essential functional step of many processes

Protein-ligand interactions

Hydrophobic effect Van der Waals interactions

Hydrogen bonds

Salt bridges

Hydrophobic effect

Saturation Binding Experiment

Main Parameters:

1. Affinity: dissociation constant (Kd)

Gdo=- RT ln(Kd)

2. Total amount of receptors (RT) or stoichiometry of the binding (n, m)

Additional information:

Type of the binding (cooperative or non-cooperative), site accessibility, site heterogeneity, etc.

Saturation Curve and Scatchard Plot

[RL]=RT [L] / (Kd+[L]) [RL] / [L] =RT / Kd - [RL] /Kd

Effects

Experimental setup

Technical considerations

1. RT<<LT, which means that [L] is close to LT

- 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 LT=2-30 M

Protein concentration?

About 0.2 M in this case

My Last Weekend’s Binding Experiment

Competitive Binding Experiment

Advantage:

Cheep

Ligand is labeled, whereas inhibitors are not –

I1, I2, I3, I4 ...

My Last Weekend’s Binding Experiment

[RL]i/[RL]=1/(1+[I]/IC50), Ki=IC50/(1+LT/Kl ), but only when RT<<LT and RT<<IT