The dissociation constant for the binding of the ligand to a GPCR is 10^-10 M. I
ID: 300139 • Letter: T
Question
The dissociation constant for the binding of the ligand to a GPCR is 10^-10 M. It was found that 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, 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 transcription factor, binds to an enhancer element of a gene and also interacts with the RNA transcription machinery 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?Explanation / Answer
a) The X-protein has a binding domain for interacting with other proteins. this interaction can be find by many techniques.
Isolate cells and provide them the condition to express the desired protein. After incubating the cells Isolate chromatin from cells, we will perform chromatin immunoprecipitation chip interaction to DNA microarray technology. This method is called as ChIP-chip method
After isolation of chromation, use adapters of enhancer DNA for isolating the desired transcription factor bound DNA.
Use enzyme specific digestion to isolate adapter linked chromatin. Now use PCR for isolated products to form large number of amplicons.
Use m-scale beads to isolate the amplicons and then immobilize them onto a surface. We use formaldehyde for immobilization.
Now use sonication method to shear the immobilized chromatin into small fragments of 200–600 bp.
Finally use antibodies to immunoprecipitate for finding out the TF-DNA complex.
b) To find out whether other proteins will interact to protein-X, we use ligand specific surface Plasmon resonance. If a protein binds to protein-X, reveal the presence of interaction by fluorescent counting.
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