Interaction Sites of Tropomyosin in Muscle Thin Filament as Identified by Site-Directed Spin-Labeling

Abstract

To identify interaction sites we measured the rotational motion of a spin label covalently bound to the side chain of a cysteine genetically incorporated into rabbit skeletal muscle tropomyosin (Tm) at positions 13, 36, 146, 160, 174, 190, 209, 230, 271, and 279. Upon the addition of F-actin, the mobility of all the spin labels, especially at position 13, 271, or 279, of Tm was inhibited significantly. Slow spin-label motion at the C-terminus (at the 230th and 271st residues) was observed upon addition of troponin. The binding of myosin-head S1 fragments without troponin immobilized Tm residues at 146, 160, 190, 209, 230, 271, and 279, suggesting that these residues are involved in a direct interaction between Tm and actin in its open state. As immobilization occurred at substoichiometric amounts of S1 binding to actin (a 1:7 molar ratio), the structural changes induced by S1 binding to one actin subunit must have propagated and influenced interaction sites over seven actin subunits.

Myosin and Other Energy-Transducing ATPases: Structural Dynamics Studied by Electron Paramagnetic Resonance

“…This may be consistent with the reports of multiple structural states and an incomplete shift to one structural state upon Ca 2+ activation for the Tm position of cardiac muscle visualized by cryoelectron microscopy [74]. [107]. Each marker color indicates the sample preparation shown on the right side of the figure.…”

“…In the three-state model, the movement of Tm occurs upon transition from the blocked to the closed state and proceeds further upon binding of the myosin heads to actin, resulting in the open state [105,106]. To identify interaction sites, Ueda et al [107] used the SDSL-EPR method and measured the rotational motion of a spin label covalently bound to the side chain of a cysteine genetically incorporated into rabbit skeletal muscle tropomyosin (Tm) at 10 positions along the entire length ( Figure 5). Upon the addition of F-actin, the mobility of all the spin labels, especially at the position joint region of Tm, was significantly inhibited.…”

“…The binding of myosin-head S1 fragments without troponin immobilized Tm residues at positions along its length, suggesting that these residues are involved in a direct interaction between Tm and actin in its open state [107]. As immobilization occurred at substoichiometric amounts of S1 binding to actin (a 1:7 molar ratio), the structural changes induced by S1 binding to one actin subunit must have propagated and influenced interaction sites over seven actin subunits.…”

“…Yamamoto et al 5.Tm -Tn-A 7 -S1 7 6.Tm -A 7 -S1 7 Figure 5. Profile of effective rotational correlation time as a function of the number of spin-labeled tropomyosin residues [107]. Each marker color indicates the sample preparation shown on the right side of the figure.…”

Clinically Divergent Mutation Effects on the Structure and Function of the Human Cardiac Tropomyosin Overlap

“…41 The assays were performed at 25 °C in 10 mM KCl, 4 mM MgCl 2 , and 20 mM Tris-HCl (pH 7.6), with 0.05 mM CaCl 2 for samples with calcium and 1 mM EGTA for samples without calcium. The protein concentrations were 4 µ M F-actin, 0.7 µ M Tm, 0.8 µ M Tn, and 1 µ M S1.…”