Abstract
A multi-site, steady-state Förster resonance energy transfer (FRET) approach was used to quantify Ca2+-induced changes in proximity between donor loci on human cardiac troponin I (cTnI), and acceptor loci on human cardiac tropomyosin (cTm) and F-actin within functional thin filaments. A fluorescent donor probe was introduced to unique and key cysteine residues on the C- and N-termini of cTnI. A FRET acceptor probe was introduced to one of three sites located on the inner or outer domain of F-actin, namely Cys-374 and the phalloidin-binding site on F-actin, and Cys-190 of cTm. Unlike earlier FRET analyses of protein dynamics within the thin filament, this study considered the effects of non-random distribution of dipoles for the donor and acceptor probes. The major conclusion drawn from this study is that Ca2+ and myosin S1-binding to the thin filament results in movement of the C-terminal domain of cTnI from the outer domain of F-actin towards the inner domain, which is associated with the myosin-binding. A hinge-linkage model is used to best-describe the finding of a Ca2+-induced movement of the C-terminus of cTnI with a stationary N-terminus. This dynamic model of the activation of the thin filament is discussed in the context of other structural and biochemical studies on normal and mutant cTnI found in hypertrophic cardiomyopathies.
Highlights
The Ca2+-tropinin complex acts as a molecular-switch in the regulation of cardiac muscle contraction
Cardiac thin filaments are composed of polymerized actin protomers decorated with stoichiometric levels of the tropomyosin dimer and the troponin complex, in the ratio of 7:1:1 respectively. cTn is composed of three subunits: troponin C (TnC), troponin I (TnI) and troponin T (TnT) [1]
In a crystal structure of the 52 kD core domain of human cardiac Tn [46], the N-terminal region of TnI, TnT and C-terminal domain of TnC constitute an ‘‘IT arm’’, that anchors the complex at the C-terminus of Tm
Summary
The Ca2+-tropinin complex acts as a molecular-switch in the regulation of cardiac muscle contraction. A recent kinetic study suggests that structural transitions are involved during two steps of Ca2+activation of the thin filament – these include the rapid dissociation of the C-terminal (mobile) domain of TnI from the actin filament, associated with Ca2+-binding, and a slower switching of the inhibitory region on TnI that is implicated in inhibiting the formation of strongly bound cross-bridges [28]. These earlier studies have for the most part been carried out on thin filament preparations derived from skeletal muscle. The acceptor sites were selected to probe motions of cTnO with respect to the outer and inner domains of Factin as well as cTm
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