Structural Dynamics of Troponin I during Ca2+-Activation of Cardiac Thin Filaments: A Multi-Site Fo ¨ rster Resonance Energy Transfer Study Hui Wang 1 , Joseph M. Chalovich 2 , Gerard Marriott 3 * 1 Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina, United States of America, 2 Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, North Carolina, United States of America, 3 Department of Bioengineering, University of California, Berkeley, California, United States of America Abstract A multi-site, steady-state Fo ¨ rster resonance energy transfer (FRET) approach was used to quantify Ca 2+ -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 Ca 2+ 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 Ca 2+ -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. Citation: Wang H, Chalovich JM, Marriott G (2012) Structural Dynamics of Troponin I during Ca2+-Activation of Cardiac Thin Filaments: A Multi-Site Fo ¨ rster Resonance Energy Transfer Study. PLoS ONE 7(12): e50420. doi:10.1371/journal.pone.0050420 Editor: Friedrich Frischknecht, University of Heidelberg Medical School, Germany Received August 8, 2012; Accepted October 23, 2012; Published December 5, 2012 Copyright: ß 2012 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health (HLO69970 awarded to GM and AR40540 to JC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: marriott1@berkeley.edu Introduction The Ca 2+ -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 (cTm) and the troponin complex (cTn), 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]. TnC, which serves as the Ca 2+ - receptor, has two globular sub-domains linked by an extended a- helix [2,3]. TnI interacts with F-actin, TnC, TnT and tropomy- osin (cTm). TnI inhibits the actomyosin ATPase in the thin filament although inhibition is reversed upon Ca 2+ -binding to the TnC complex [4]. High-resolution structural analyses have shown that the two EF-hand motifs on the N-lobe of skeletal TnC open up upon Ca 2+ -binding [5–8] leading to the unfolding of a short a- helix within the TnI inhibitory segment [9]. This conformational change allows TnI to bind more tightly to TnC [10,11] and, as a result, the interaction between TnI and F-actin weakens. This latter change is key to the activation of the thin filament, as it should allow myosin to form a strong bond with F-actin [12,13]. Collectively, these studies suggest that the activation of the thin filament requires significant, Ca 2+ -triggered, concerted structural dynamics [14]. Understanding the role of protein structural dynamics in Ca 2+ - regulation of muscle contraction might help to explain how specific single point mutations in thin filament proteins lead to hypertrophic and dilated cardiomyopathies (HCM and DCM). For example, many of the HCM mutations in TnC and TnI that lead to reduced cardiac output are single point and conservative [15–19], and on the face of it, unlikely to alter the overall structure or interactions of the mutated protein within the thin filament. We are testing the hypothesis that the deleterious effects of these mutations are a result of altered structural dynamics, for example one that might change the rate of a conformational transition or coupling to a neighboring subunit in the filament. Ideally one would test this hypothesis by carrying out high-resolution structural analyses of individual Tn subunits within functional thin filaments at different states of the crossbridge cycle. Cryo- electron microscopy of reconstituted or intact muscle fibers is the most informative of these techniques although the information is carried out on fixed samples. On the other hand, Fo ¨rster resonance energy transfer (FRET) provides sensitive and dynamic information on changes in proximity between specific loci on multiple labeled proteins within functional thin filament under physiological conditions of temperature and ionic composition. The FRET-approach has been applied in the characterization of proximity relationships within or between Tn subunits and their PLOS ONE | www.plosone.org 1 December 2012 | Volume 7 | Issue 12 | e50420