Question

You are characterizing the binding of a ligand to a protein. Knowing that the rate of association between the protein and ligand is diffusion controlled and that the K_D is expected to be around 100 µM,

a) What do you expect that half-life of the complex to be?

b) What method would you choose to measure the K_D? Briefly describe why the method is appropriate

Answer 1

To characterize the binding of ligand to protein, measurement of association rate constant (K₁) and dissociation rate constant (K_-1) needed to estimate the equilibrium dissociation constant (KD)

K_D = (K_-1/K₁)        ---------------------(1)

a) Half life of the protein - ligand complex (T_1/2) = 0.693/K_-1   ----------------------------(2)

Given, K_D = 100µM,

if diffusion controlled association K₁≈10⁷ M^-1 sec^-1 ------------------(3)

substitute (3) in (1)

K_-1 =    100 x 10 ^-6 M x 10⁷ M^-1 sec^-1

K_-1= 1000 sec^-1           ---------------------(4)

substitute (4) in (2)

Half life of the complex expected to be 7 msec.

(b) Brownian dynamics simulations method used to calculate the diffusion-controlled rate constant k_D. Because this method easy to comphrend for different protein shapes and various interaction potentials. In compared to analytical theories, have structural assumption are simple for evaluate transient complexes.