Tropomyosin dynamics in cardiac thin filaments: A multi-site Foerster resonance energy transfer and anisotropy study

¹ University of Wisconsin
² East Carolina University Medical School

^* To whom correspondence should be addressed. E-mail: marriott{at}physiology.wisc.edu.

Submitted on September 1, 2007
Revised on October 30, 2007
Accepted on 15 January 2008

	Abstract

Cryo-electron microscopy studies have identified distinct locations of tropomyosin (Tm) within the Ca²⁺-free, the 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. Multi-site FRET and fluorescence anisotropy analyses showed that S1-binding to Ca²⁺-thin filaments triggered a uniform displacement of cTm towards 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. While 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.

Key Words: FRET, thin filament, tropomyosin