L. Šimunová1, 3, Z. Lánský1, 2, M. Kubala5, J. Teisinger1 and E. Amler3, 4


1Institute of Physiology, Academy of Sciences of Czech Republic

2Institute of Physics, Charles University, Czech Republic

3Institute of Biophysics of the 2nd Medical School, Charles university, Czech Republic

4Institute of Experimental Medicine, Academy of Sciences of Czech Republic

5Department of experimental physics, Faculty of Science, Palacky University, Czech Republic


The ATP binding site of the Na+/K+-ATPase is located on the large cytoplasmatic loop (H4-H5 loop) of the alpha-subunit. Conformational changes of the H4-H5 loop has been observed upon the ATP binding as well as the Na+/K+-ATPase association in the presence of MgATP. In this study we have analyzed the changes in conformation and quartery structure of the H4-H5 loop by the means of site-directed mutagenesis and fluorescence spectroscopy. DNA sequence of a large cytoplasmatic loop of Na+/K+-ATPase alpha-subunit was subcloned into expression vector pET28b and expressed as a (His)6 - tag fusion protein in E. coli BL21 cells. We have prepared a set of single-tryptophan mutants mapping the surface of the H4-H5 loop. Mg, MgATP, NaATP titration experiments were performed with these point mutants and the fluorescence emission was used to monitor changes of polarity in the tryptophan microenvironment caused by the substrate binding, thus monitoring the changes of the protein conformation. Time resolved fluorescence measurements were performed with selected point-mutants to specify more closely the effects of the ligand binding on the protein conformation and changes in quartery structure. Results of the fluorescence experiments were thereafter interpreted in terms of previously released computer model of the H4-H5 loop [1].


This work was supported by Centre of Neurosciences LC554 of the Ministry of Education, Youth and Sports of the Czech Republic, GAUK (No. 200053), GACAS (T400110403), and Grant Agency of Czech Republic project (No. H148).


1. Ettrich R. et al., J Mol Model, 7 (2001) 184 -192.