The dissociation constant for the binding of the ligand to a

The dissociation constant for the binding of the ligand to a GPCR is 10^-10 M. It was found that ligand binding to only 10% of the around 1000 GPCRs on the cell is required for the maximal activation of the G protein. Calculate what is the ligand concentration required to bring about the maximal activation. Now you mutate the G-protein alpha_s subunit to increase its affinity to GTP. What will be the effect on ligand binding to the receptor and why? If these mutant G-proteins are now treated with a non-hydrolyzable analog of GTP, what is going to happen to the ligand binding and why? Design an experiment to find out whether a protein X, which you suspect is a inscription factor, binds to an enhancer element of a gene and also interacts with the RNA transcription machines to activate transcription. You have done a structural analysis of X and have found that it is predicted to have a domain that binds to DNA and another domain which binds to protein. How can you find out other cellular proteins which might interact with protein X? Design an experiment to find out whether the binding of a ligand to a GPCR causes the dissociation of the G_alpha subunit of the G protein from the G_beta_gamma subunit. What will happen if you do the same experiment in presence of excess GDP?

Solution

3 (a) Yes, Binding of a ligand to GPCR dissociates the G alpha subunit from G beta gamma subunit. When an agonist binds to GPCR, it stimulates the conformational changes in the active GPCR which is transmitted to Galpha present in the molecule. When G alpha subunit gets activated, it starts exchanging GTP instead of GDP and hence G alpha gets dissociated from G beta gamma part.

In presence of excess GDP, the dissociation will not be so spontaneous. It may get delayed.