Key Points
- To measure the association constant between myosin subfragment-1 and F-actin using a novel time-resolved fluorescence depolarization technique.
- Capitalized on differences in rotational mobility between free myosin subfragment-1 (S-1) and F-actin.
- Uncoupled the time dependence of anisotropy decay from association/dissociation kinetics to fit decay curves to a two-term model weighted by free and bound S-1 mole fractions.
- Conducted binding measurements at 4 °C, an ionic strength of 0.16 M, and pH 7.0.
- The association constant (Ka) of S-1 for F-actin was determined to be (1.73 ± 0.35) × 10⁶ M⁻¹ at infinite dilution.
- The standard free energy (-ΔG°) of F-actin binding to S-1 was found to be quantitatively similar to that of ATP binding to S-1.
Structured PICO
PPopulationMyosin subfragment-1 (S-1) and F-actin (in vitro model)
IInterventionTime-resolved fluorescence anisotropy measurement
OOutcomeAssociation constant (Ka) for myosin S-1 and actinsurrogate
The study demonstrates that the free energy of binding of F-actin to S-1 is similar to that of ATP to S-1, providing insights into the biophysics of muscle contraction.