Different phosphorylation states of native titin filaments visualized by atomic force microscopy

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 structural alterations due to phosphorylation, single-molecule experiments must be carried out on individual titin molecules, where the effect of phosphorylation can be tested individually. In our work, we isolated individual titin molecules from rabbit and mouse skeletal muscle. The isolated native titin molecules showed high levels of phosphorylation, when stained with phosphoprotein gel stain. The isolated molecules were treated with lambda protein phosphatase to decrease the in situ phosphorylation level of the polymer. To investigate the effect of the treatment on titin's structure, we visualized surface-bound titin molecules by atomic force microscope and found that the C-terminal region of dephosphorylated titins collapses into a compact, coiled structure. Our findings suggest that the structure of the A-band region of titin is highly sensitive for phospohrylation which might play a role in controlling protein-titin interactions in the A-band.