Tropomyosin dynamics in cardiac thin filaments: A multisite Forster resonance energy transfer and anisotropy study

Cryoelectron microscopy studies have identified distinct locations of tropomyosin (Tm) within the Ca2+-free, Ca2+-saturated, and myosin-S1-saturated states of the thin. lament. On the other hand, steady-state Forster resonance energy transfer (FRET) studies using functional, reconstituted thin. laments under physiological conditions of temperature and solvent have failed to detect any movement of Tm upon Ca2+ binding. In this investigation, an optimized system for FRET and anisotropy analyses of cardiac tropomyosin (cTm) dynamics was developed that employed a single tethered donor probe within a Tm dimer. Multisite FRET and fluorescence anisotropy analyses showed that S1 binding to Ca2+ thin. laments triggered a uniform displacement of cTm toward F-actin but that Ca2+ binding alone did not change FRET efficiency, most likely due to thermally driven fluctuations of cTm on the thin. lament that decreased the effective separation of the donor probe between the blocked and closed states. Although Ca2+ binding to the thin. lament did not significantly change FRET efficiency, such a change was demonstrated when the thin. lament was partially saturated with S1. FRET was also used to show that stoichiometric binding of S1 to Ca2+-activated thin. laments decreased the amplitude of Tm fluctuations and revealed a strong correlation between the cooperative binding of S1 to the closed state and the movement of cTm.