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The Impact of Nε-Acryloyllysine Piperazides on the Conformational Dynamics of Transglutaminase 2

Heerwig, A.; Kick, A.; Sommerfeld, P.; Eimermacher, S.; Hartung, F.; Laube, M.; Fischer, D.; Pietzsch, H.-J.; Pietzsch, J.; Löser, R.; Mertig, M.; Pietsch, M.; Wodtke, R.


In addition to classic functions of proteins such as acting as biocatalyst or binding partner, the conformational states of proteins and their remodeling upon stimulation needs to be considered. A prominent example that undergoes comprehensive conformational remodeling, is transglutaminase 2 (TGase 2), whose distinct conformational states are closely related to particular functions. Its involvement in various pathophysiological processes, including fibrosis and cancer, motivates the development of theranostic agents, particularly based on inhibitors that are directed towards the transamidase activity. In this context, the ability of such inhibitors to control the conformational dynamics of TGase 2 emerges as an important parameter, and methods to assess this property are in great demand. Herein, we describe the application of the switchSENSE® principle to detect conformational changes caused by three irreversibly binding Nε-acryloyllysine piperazides, which are suitable radiotracers candidates of TGase 2. The switchSENSE® technique is based on DNA levers actuated by alternating electric fields. These levers are immobilized on gold electrodes with one end, and at the other, distal end of the lever, the TGase 2 is covalently bound. A novel computational method is introduced for describing the resulting lever motion to quantify the extent of stimulated conformational TGase 2 changes. Moreover, as a complementary biophysical method, native polyacrylamide gel electrophoresis was performed under similar conditions to validate the results. Both methods prove the occurrence of an irreversible shift in the conformational equilibrium of TGase 2, caused by the binding of the three studied Nε-acryloyllysine piperazides.

Keywords: transamidase inhibitor; DNA nanolever; binding kinetics; conformational change

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