Poster Presentation 2014 International Biophysics Congress

Reactivity of cysteins on the large cytoplasmic segment (C45) of Na+/K+-ATPase (#327)

Jaroslava Geleticova 1 , Miroslav Huliciak 1 , Martin Kubala 1
  1. Palacky University, Olomouc-Holice, Czech Republic

Na, K-ATPase (NKA) is a plasma membrane protein responsible for maintaining the membrane potential by actively transporting sodium and potassium ions, and its proper function  is essential for all animal cells. Large cytoplasmatic segment connecting transmembrane helices 4 and 5 (C45) is soluble and can be overexpressed in E.coli with high protein yields. Up to now, all experiments suggested that the structure of isolated C45 is not altered, making it ideal model system for interaction studies with soluble ligands.

Many ligands specifically interact with sylfhydryl groups of cysteinyl residues, however, the reactivity of each cystein can be influenced by its accessibility from solvent or interactions with adjacent amino acids..

The reactivity of cysteins on isolated C45 was assessed by sulfhydryl-reactive species and using site-directed mutagenesis, we prepared a set of cysteine mutants of the C45 segment (C367S, C421S, C452S, C511S, C456S, C456S+C457S, C549S, C577S, C599S, C656S and C698S) to reveal site-specific interactions.

Acknowledgments

This work was supported by grant OPVK, CZ.1.07/2.3.00/20.0057 from Czech Ministry of Schools, Youth and Sports, by grant LO1204 from the National program of Sustainability and from the grant IGA_PrF_2014_029 from Palacky University in Olomouc.