Molecular Sciences I. (Poster discussion will take place in the Aula during the Coffee Break)
Andrea Balogh-Molnár1, Dominik Sziklai1, Zsolt Mártonfalvi1
1 Semmelweis University, Department of Biophysics and Radiation Biology
During muscle stretch, elastic or "passive" force develops which is mainly determined by the giant protein titin that forms the third filament system of muscle sarcomeres. The magnitude of this force is mainly dependent on the elasticity of the titin filaments, which is primarily determined by the structure of the polypeptide chain. However, it is suggested that post-translational modifications of titin, such as phosphokinase activity, regulate the sarcomeric passive force development. Mechanical studies on single myofibrils revealed that different protein kinases alter the passive tension of muscle, surprisingly, in opposite ways. This suggests that phosphorylation of sarcomeric proteins by various kinases is an essential regulatory mechanism of passive force. However, the extrapolation of these findings to titin’s phosphorylation has not been studied so far at the single-molecule level. To reveal titin’s contribution to the alterations of passive force due to phosphorylation, single-molecule experiments must be carried out on individual titin molecules, where the effect of each relevant phosphorylation state can be tested individually. In our work, we isolated individual full-length titin molecules from rabbit skeletal and cardiac muscle. The isolated native titin molecules showed high levels of phosphorylation, when stained with phosphoprotein gel stain. The isolated molecules were treated with various phosphatases and kinases to alter the in situ phosphorylation state of the polymer. To investigate the effect of the various phosphorylation states on titin's structure, we visualized surface-bound titin molecules by atomic force microscope and measured contour length to end-to-end length ratios of titin polymers.
This work is founded by the NKFIH FK128956 grant.