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

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)

Joseph M. Chalovich (Creator)

Shu Mao (Creator)

Gerard Marriott (Creator)

Hui Wang (Creator)

Institution

East Carolina University (ECU )

Web Site: http://www.ecu.edu/lib/

Abstract: Cryoelectron microscopy studies have identified distinct locations of tropomyosin (Tm) within the Ca21-free Ca21-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 Ca21 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 Ca21 thin filaments triggered a uniform displacement of cTm toward F-actin but that Ca21 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 Ca21 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 Ca21-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. Originally published Biophysical Journal Vol. 94 No. 11 June 2008

Additional Information

Publication

Other

Biophysical Journal. 94:11(June 2008) p. 4358-4369.

Language: English

Date: 2011

Keywords

cryoelectron microscopy, cardiac thin filaments, Tropomyosin

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