Question

You are studying the role of dynamin in the budding and pinching off mechanism of clathrin coated vesicles from the plasma membrane. You know that dynamin uses GTP hydrolysis to perform its function, however you would like to test how dynamin behaves in the presence of a non-hydrolyzable analog of GTP. Describe an experiment that allows you to observe the function of dynamin in the presence of either GTP or a non-hydrolyzable GTP analog. What do you predict will happen in the presence of the analog?

Answer 1

Dynamin refers to a GTPase protein, which plays an essential function in hydrolyzing GTP. To monitor the function of dynamin, a homogenous bioluminescent assay, that is, a GTPase-Glo Assay can be used to determine the working of GTPases and the proteins associated with it.

This assay helps in quantifying the GTP left post a GTPase reaction. The leftover GTP gets converted into ATP by supplementation of the GTPase-Glo reagent to the assay. This then gets transformed into a luminescent signal by the supplementation of the detection reagent.

In case if dynamin is working and is present in an active state, then there will be less or no availability of GTP, and thus, detection of luminescence will not be done. On the other hand, in the presence of a non-hydrolyzable GTP, the conversion of GTP into ATP will take place, as dynamin fails to hydrolyze it, and thus, detection of luminescence will be done.