THE PURIFICATION PROCESS OF TRPC6 C-TAIL AND CALMODULIN- OPTIMIZATION FOR CRYSTALLIZATION

B. Holakovská, L. Gryčová, H. Janoušková, J. Bílý, J. Teisinger

Institute of Physiology, Academy of Sciences of the Czech Republic

TRP (transient receptor potential) channels represent multifunctional sensors perceiving wide spectrum of environmental cues in form of physical and chemical stimuli. They are widely expressed in the CNS and peripheral cell types. They are involved in numerous fundamental cell functions. Recently, many pathological conditions have been linked to TRP dysfunctions. However many aspects of physiology and regulation of TRPs are still elusive.

It has been proved that TRPC6 plays an important role in vascular and pulmonary smooth muscle cells and it has been proposed to play a critical role in the intravascular pressure-induced depolarization and constriction of small arteries and arterioles.

Unveiling the structure of complex of TRPC6 C-tail with calmodulin could contribute to elucidation of mutual structural adjustment of these two molecules in space, their mutual interactions on molecular level. Detailed studies of the structure of this complex could help with prediction of therapeutically significant substances applicable in clinical practice for treatment of diseases caused by TRP dysfunctions.

Purification process of recombinant TRPC6 C-tail and calmodulin was suggested. Both proteins were expressed in E. coli expression system. The cells were resuspended, digested with lysozyme and sonicated. The TRPC6 C-tail peptide was obtained by successive affinity chromatography on HisPur resin column and gel filtration. Calmodulin was purified on the column of CL4B-Sepharose and gel filtration. The proteins were mixed together and refined to a high purity by gel filtration. The sample was further used for crystallization experiments using the method of “hanging drop “.

This work was supported by Grant GAAV IAA600110701, GACR 303/07/0915, project (No. H148), Centre of Neurosciences No.LC554 MSMT CR, Research project No. AV0Z.