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Tropomyosin Dynamics in Cardiac Thin Filaments: A Multisite Förster Resonance Energy Transfer and Anisotropy Study

Hui Wang ^*, Shu Mao ^*, Joseph M. Chalovich  and Gerard Marriott ^*

^* Department of Physiology, University of Wisconsin, Madison, Wisconsin 53706; and Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, North Carolina 27834

Correspondence: Address reprint requests to Gerard Marriott, Dept. of Physiology, University of Wisconsin, Madison, 1300 University Ave, Madison, WI 53706. Tel.: 608-262-6309; E-mail: marriott{at}physiology.wisc.edu.

Cryoelectron microscopy studies have identified distinct locations of tropomyosin (Tm) within the Ca²⁺-free, Ca²⁺-saturated, and myosin-S1-saturated states of the thin filament. On the other hand, steady-state Förster resonance energy transfer (FRET) studies using functional, reconstituted thin filaments 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 filaments 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 filament that decreased the effective separation of the donor probe between the blocked and closed states. Although Ca²⁺ binding to the thin filament did not significantly change FRET efficiency, such a change was demonstrated when the thin filament was partially saturated with S1. FRET was also used to show that stoichiometric binding of S1 to Ca²⁺-activated thin filaments 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.