Dephosphorylation-induced structural reorganization of skeletal muscle titin molecules

During muscle stretch, an elastic or "passive" force develops, primarily determined by the giant protein titin, which forms the third filament system of the muscle sarcomere. The magnitude of this passive force depends largely on the elasticity of titin, governed by the structure of its polypeptide chain. In addition to its structural role, post-translational modifications-particularly phosphorylation by protein kinases-have been suggested to modulate sarcomeric passive force. Mechanical studies on single myofibrils have shown that different kinases can alter passive tension in opposing directions, highlighting phosphorylation as a critical regulatory mechanism. However, the direct contribution of titin phosphorylation to these effects has not been investigated at the single-molecule level. To address this, we conducted single-molecule experiments on individual titin molecules isolated from the m. longissimus dorsi skeletal muscle of rabbit. Phosphoprotein gel staining revealed that the native molecules were highly phosphorylated. To manipulate phosphorylation status, the molecules were treated with λ-protein phosphatase, thereby generating a hypophosphorylated state. Atomic force microscopy of surface-bound titin filaments revealed a marked conformational change in response to dephosphorylation: the C-terminal region of titin collapsed into a compact, coiled structure. These findings suggest that phosphorylation influences titin's nanomechanical structure and may play a direct role in regulating passive muscle tension.